ALBERT

Description: In myeloma, plasma exchange (PE) has been suggested to prevent rapidly progressive kidney failure by reducing exposure to nephrotoxic light chains. We carried out a randomized controlled multi-centre trial comparing PE or no PE in 104 patients of whom 101 met the inclusion, exclusion criteria and 4 were lost to follow-up. We compared baseline characteristics as well as renal outcomes and performed a futility analysis to determine the sample size necessary for potential statistical significance for the changes noted. Thirty-nine patients were randomized to the control group and 58 to the PE group with a 6-month follow-up. The baseline characteristics of these 2 groups were similar including serum creatinine, dialysis dependence, age, gender, serum calcium, serum albumin, 24 -hour urine for protein levels and Durie-Salmon myeloma staging. Thirteen (33.3%) of the control group and 19 (33.3%) of the PE group died within 6 months of follow up. Ten patients (31%) in the control and 10 patients (21%) in the PE arm were dialysis dependent at 6 months. Seven patients (47%) came off dialysis in the control and 13 patients (59%) in the PE arm with the mean number of dialysis days from 0–6 months being 45.7±67.6 in the control versus 29.2±56.1 in the PE arm at 6 months. The mean serum creatinine in the control group was 314.6±256.1 μmol/L versus 215.4±215.3 μmol/L in the PE group and the composite end point of death, dialysis or serum creatinine 〉254 μmol/L occurred in 12 (30.8%) in the control and 11 (19.3%) in the PE arm. The futility analysis to indicate the per group sample size necessary to achieve statistical significance at 6 months for the difference we observed was infinite for cumulative mortality, 805 for dialysis dependence, 2418 for coming off dialysis, 321 for number of dialysis days, 132 for creatinine difference of 100 μmol/L and for the composite outcome of death, dialysis or creatinine〉354 μmol/L, 737. We did not observe a statistically significant difference in mortality or renal morbidity for PE versus no PE in patients with myeloma and rapidly progressive kidney failure.

Description: Objective To explore the regimen related toxicity (RRT) and the effects of super-intensified conditioning regimen combined with inducing graft versus leukemia (GVL) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for refractory leukemia which can’t obtain complete remission (CR) pre- transplantation. Methods 18 patients who did not obtain CR before transplantation received super-intensity conditioning regimen protocol(experimental group), and 62 patients with acute leukemia who obtained CR or chronic myeloid leukemian (CML) who were in the chronic phase before transplantation received total body irradiation (TBI) plus cyclophosphamide (CTX) or modified BuCY (hydroxyurea, busulfan, Ara-C, CTX) protocol (control group). Cyclosporin A was reduced rapidly and gradually or donor lymphocytic infusion (DLI) was used to induced GVL if acute graft versus host disease (GVHD) did not happened in patients with refractory leukemia at 30 days post-transplantation. The incidence and mortality of RRT during transplantation, and the rate of CR, GVHD and leukemia relapse after transplantation was investigated. Kaplan-Meier survival analysis model was used to estimate the disease-free survival (DSF) rate at 3 years post-transplantation. Results Except one patient in experimental group and two patients in control group died of transplant-related complications, all the other patients obtained hematopoietic reconstitution.’ the total RRT incidence were both 100% in two groups. The RRT of stomach intestine were most common in all the organs and the RRT incidence of experimental group and control group was 83.3% and 85.5%, respectively, in stomach intestine. The RRT incidence was 44.4% and 62.9% in oral cavity and 16.7% and 33.9% in bladder, respectively, in the experimental group and control group. There was no significance and P value was 0.823, 0.172 and 0.244, respectively, in the RRT incidence of stomach intestine, oral cavity and bladder between the two groups. The RRT mortality was 0 and 5%, respectively, and was not different (P=0.341) in the experimental and control group. Except one patient died of infection, all the other patients obtained CR in patients who were treated with supper-intensified conditioning regimen. The incidences of acute or chronic GVHD were 58.8% and 40.0% or 92.6% and 55.8%, respectively, in the experimental and control group. The incidence of leukemia relapse was 11.8% and 18.3%, respectively, in the two groups. The DSF at 3 years after transplantation was 61.2±12.3% and 65.0±7.4% (P=0.6311), respectively, in the two groups. Conclusion The consecutive super-intensified conditioning regimen combined with inducing GVL post transplantation protocol can increase the rate of CR and DFS, and dose not increase RRT incidence and mortality in allo-HSCT for the refractory leukemia which can’t obtain CR pre- transplantation.

Description: Allogeneic hematopoietic transplantation following nonmyeloablative preparative conditioning (also know as “mini-dose transplantation”) is frequently applied to patients who, on the basis of prior therapy, organ dysfunction, or co-morbid conditions may be considered ineligible for full, dose-intenstive treatment. This approach assumes that early regimen-related toxicity can be averted by the nonmyeloablative approach. We present the results obtained in a group of consecutive, unselected older adults with advanced hematologic neoplasia who underwent allogeneic transplantation from fully histocompatible siblings or unrelated donors after dose-reduced preparative conditioning in a single center. Thirty-four patients, median age 57 years (range 26–66) underwent allogeneic transplantation from siblings (14 patients) or unrelated donors (20 patients) following preparative conditioning consisting of chemotherapy only (typically fludarabine, cytarabine, and cyclophosphamide) or those drugs plus antithymocyte globulin, respectively. One recipient of a related allograft and one recipient of an unrelated allograft received preparative fludarabine/melphalan and busulfan/fludarabine/ATG, respectively. Diagnoses prior to transplantation included advanced acute leukemia and myelodysplasia (17 patients), nonHodgkin’s or Hodgkin’s lymphoma (11 patients), and CLL or Multiple Myeloma (6 patients). Allogeneic transplantation followed previous autologous transplantation in 17 patients. Time from previous autologous transplantation to nonmyeloablative allogeneic transplantation varied. Prior autograft conferred a significant adverse risk on recipients of allogeneic transplants following nonmyeloablative conditioning. Only two of 17 patients with a history of prior autograft enjoyed long-term survival as opposed to seven of 17 who had not undergone prior autologous transplant. Mortality before day + 100, typically attributed to treatment-related toxicities, occurred in 11 patients; nine of these patients had had prior autologous transplantation. Prior autologous transplantation typically was done for acute myelogenous leukemia in first complete remission and referral for allotransplant may have been an indicator of more aggressive disease biology. In conclusion, allogeneic transplantation following a nonmyeloablative preparative regimen may be associated with significant risk of early mortality in patients who undergo this treatment after prior autologous transplantation, especially for acute leukemia, suggesting that criteria for allogeneic transplantation after nonmyeloablative condition should take this risk into account. Further studies from multiple centers will be required to confirm this observation.

Description: The recovery of viable CD34+ cells reinfused into patients at the time of autologous or allogeneic transplantation is clinically an important variable, which can determine graft success or failure. In this study we analyse the recovery of viable CD34+ cells /kg pre and post cryopreservation on a total of 86 autologous stem cell products from adult and paediatric patients as well as 4 cryopreserved stem cell products from allogeneic donors. CD34 enumeration was performed on all samples pre and post cryopreservation using a novel in-house no-lyse CD34 assay (previously described ASH 2003 abstract no.1685). Cells were labelled with CD45, CD34 and 7AAD in TRUCOUNT tubes using a modified single platform ISHAGE protocol. The absolute number of viable CD34 + cells per Kg was determined. For the 77 PBSC harvest samples the mean viable CD34+ cell count was 6.0 x10^6/Kg (range 0.3 – 25.2 x 10^6/Kg) before freezing. For post thaw samples the mean viable CD34+ cell count was 5.5 x 10^6/Kg (range 0.2 – 24.6 x 10^6/Kg). The median recovery was 95% (range 48–124%). This represents a median loss post freeze/thaw of 5%. Further analysis showed a median recovery of 90% for NHL (range 48–119%, n=34), 87% for MM (range 56–115%, n=12), 92.5% for acute leukaemia (range 71–124% n=8) and 97% for non-hematological malignancies (range 50–120% n=21). There was no significant difference in the recovery of viable CD34+ cells within the four groups of malignancies (p〉0.17 for all groups tested). Similarly, autologous bone marrow collections (n=9) also showed a good recovery of viable CD34+ cells post thaw. The median viable CD34+ cell count was 8.1x10^6 /Kg (range 0.6–30.3x10^6/kg) pre-cryopreservation, compared to a median viable cell count of 6.5 x10^6/Kg CD34+ cells (range 0.6–26x10^6/Kg) post thaw, this represents a median recovery of 90% viable CD34+cells from autologous bone marrow collections. There was no significant difference in the recovery of viable CD34+ cells from autologous PBSC harvests and autologous bone marrow collections (p=0.169). We also compared the recovery of viable CD34+ cells post thaw between adult and pediatric stem cells collections. The median recovery of viable CD34+ cells from 56 adult stem cell products post thaw was 91% (range 48–120%), compared to a median recovery of 96.5% (range 50–124%) viable CD34+ cells from 30 pediatric stem cell products (p=0.06). Interestingly the greatest loss occurred in allogeneic donors, where viable CD34+ counts on fresh samples averaged 5.7 x 10^6/Kg (range 3.1–11.8 x 10^6/Kg, n=4), whereas post freeze/thaw averaged 2.2 x 10^6/Kg (range 1.2–3.3 x 10^6/Kg). Representing a mean loss of 58% of CD34+ cells. Twenty-nine patients were transplanted with a median number of 3.8x10^6 viable CD34+ cells per Kg (range 1.8–18.4x10^6/Kg), The median time to neutrophil and platelet engraftment was 12 days (range 10–18) and 14 days (range 8–65) respectively. Assaying the viability of CD34+ cells post cryopreservation may identify patients at risk of poor haematological recovery that could benefit from further stem cell collections.

Description: Isolated chronic thrombocytopenia (CT) following allogeneic HCT is associated with an increased risk of transplant-related mortality and a reduction in long-term survival. Although the pathogenesis of CT is not entirely clear, it has been speculated that immune activation and cytokine dysregulation characteristic of chronic GVHD (CGVHD) play a similar role in the pathogenesis of this disorder. To better understand the impact of endogenous cytokine expression on platelets, we analyzed pro and anti-inflammatory cytokines in the serum of patients with CT following allogeneic HCT, comparing levels to healthy donors and to patients with normal platelet counts after allo-transplantation. Serum was obtained from 3 patient groups; Group 1- healthy donor controls (n=12): Group 2- post-transplant from allogeneic HCT patients with normal platelet counts (n=31): Group 3- post transplant from patients with CT (n=11). Using micro sphere-based Luminex flow cytometry, serum was analyzed for levels of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α as well as Th(1) cytokines IL1-b, IL-2, IL-12, IFN-g and Th(2) cytokines IL-4, IL-5, and IL-10. Results: Both post transplant cohorts (Group 2 and 3) had higher mean levels of GM-CSF (110 and 120 pg/ml), IFN-g (17 and 16 pg/ml), and IL-12 (318 and 202 pg/ml) compared to their respective non-transplant (Group-1) controls (GM-CSF 23 pg/ml: p

Description: Despite significant improvement in the outlook for newly diagnosed patients with multiple myeloma (MM) the disease remains incurable. Novel therapeutic approaches are therefore required. We have previously demonstrated in vitro that both the third generation nitrogen-containing bisphosphonate zometa and the HMG-CoA reductase inhibitor fluvastatin have anti-proliferative and apoptosis-inducing effects against human myeloma cell lines (HMCL). Furthermore, isobologram analyses with the 2 compounds have demonstrated synergistic activity against HMCL. Based on this we have undertaken a pilot study of fluvastatin and zometa (FluZom) in a group of patients with progressive plasma cell disorders (PCD). A 16-week treatment phase was planned with patients commencing fluvastatin 40mgdaily on day 1 escalating to 80mg daily from day 15 to day 112. Zometa 4mg IV over 15 minutes was given on days 29, 57, 85 and 112. Any patient demonstrating any response, based on conventional criteria, or stable disease (SD) by day 112 remained on treatment until further progression (PD). PD prior to day 112 mandated treatment withdrawal. Eleven patients were enrolled on trial - progressive MM (n = 9), smouldering myeloma (SMM) progressing to MM (n = 1) and progressive POEMS disease with multisystem involvement (n = 1). All except the SMM patient were heavily pretreated - median 3 (range, 0 – 6) prior treatment regimens, 7 with prior ASCT and 4 with prior radiotherapy. Median age was 61 years (range, 44 – 74). Six patients completed 16 weeks of therapy, 5 progressed prior. Of the former, 4 (36%) demonstrated SD and continued on therapy to a total of 20, 23 (patient no. 2), 28 (patient no.1) and 45 weeks. No patient demonstrated a disease response based on standard criteria. Patients no.’s 1 and 2 were pancytopenic prior to therapy and demonstrated normalisation of haemoglobin (no. 1), platelets (no. 1) or total leucocytes (no.’s 1 and 2). Two patients, neither of whom achieved SD but who completed 16 weeks of therapy, had a plasma cell labelling index (PCLI) undertaken pre and post therapy, both showed a reduction in the PCLI following therapy - 5.4% to 3.6% and 2.6% to 1.3%, respectively. Therapy was well tolerated with 3 patients developing transient elevation of liver transaminases. We conclude that FluZom while not producing disease response as judged by conventional criteria did nonetheless exhibit beneficial biological activity in a minority of this cohort of progressive PCD patients. Further evaluation in patients with early and/or untreated disease is warranted.

Description: Backgound: The clinical course of multiple myeloma is often associated with significant bone related morbidity. This can be modified by the use of bisphosphonates. Studies have shown renal toxicity associated with different bisphosphonates especially following the exposure of zoledronic acid. Published literature suggests renal function deterioration occurs in 8.8–15.2% of patients at recommended dose of 4 mg infused intravenously over 15 minutes. Methods: We retrospectively reviewed the records of all patients with multiple myeloma who received bisphosphonates during the period of January 2002-June 2004 at our institution. 114 patients were analyzed (male/female 63/51; age-median 69;mean 71;range 40–92). 61 (54%) were 〉70 years of age. They received a total of 1301 doses (mean 11.4) during this period. The type of bisphosphonate used was: zolendronate: 58; pamidronate: 23; pamidronate changed to zolendronate (both) : 33. Patients were categorized to the type and sequence of bisphosphonates [ pamidronate vs. zoledronate and pamidronate followed by zoledronate (both) ], age and sex. Renal dysfunction was defined as an increase in serum creatinine of 〉0.5 mg/dl over baseline. Results: There were 19 patients (16.7%) who developed renal dysfunction. 15 of the 19 episodes (79%) occurred in the 70 years and older group. The table shows the distribution of patients, type of bisphosphonate and the distribution of patients with renal toxicity. Conclusion: This analysis showed increase in renal dysfunction occurs in all ages with use of bisphosphonates. The elderly may be particularly susceptible to this toxicity. Although we have not analyzed the impact of associated comorbidities (including type of multiple myeloma) leading to renal insufficiency in this study, the elderly patients may need more close monitoring of renal function with the use of bisphosphonates. Age and renal impairment with bisphosphonate use Age Zolendronate Pamidronate Both Total Number in paranthesis indicates the renal impairment case 40–49 6 (0) 2 (0) 2 (0) 10 (0) 50–59 10 (0) 1 (0) 4 (0) 15 (0) 60–69 14 (2) 3 (0) 11 (1) 28(3) 70–79 19 (3) 9 (1) 14 (4) 42 (8) 80+ 9 (3) 8 (0) 2 (4) 19 (7) Total 58 (9) 23 (1) 33 (9) 114 (19)

Description: Background: Nonmyeloablative conditioning regimens are more and more widely used in the setting of stem cell transplant due to a reduced transplant related mortality. However, these conditioning regimens include more immunosuppressive agents and little data in terms of immunological recovery are found in the literature. Population: 24 patients (pts) admitted for nonmyeloablative transplant from HLA identical donors were prospectively followed during a period of 1 year. All pts received a conditioning regimen consisting of rabbit ATG (fresenius), Fludarabine phosphate, Cyclophosphamide ( ARA-C for myeloid malignancies) cyclosporin A and mycophenolate mofetil (MMF). Lymphocytes markers expression as well as lymphocytes absolute numbers were studied to evaluate the cellular immunologic recovery. IgG, IgA and IgM levels were also monitored during one year. Pts were stratified according to the presence of GVHD(acute and chronic) or not. Results: 24 pts with a median age of 52 (20–64) years old transplanted with an attenuated conditioning regimen, were evaluated. Median delay to recover 500 ANC and 20000 plt/ul was 8 days. Without GVHD, the pts recover a normal level of CD3, CD8, CD19,CD56 and CD45RO positive lymphocytes within 6 months. CD4+ and CD45RA + lymphocytes did not reach normal values within 1 year; Therefore the CD4/CD8 ratio remains low for more than 12 months. This observation explains the high incidence of oppotunistic infections in these pts even 6 months after transplantion. In our small seies,with or without GVHD neither IgA, nor IgM recovered within 1 year. IgG recovery is not evaluable because of human IVIG administration monthly after transplant. Conclusion: our small series confirms the rapid hematologic recovery after nonmyeloablative transplant.Cellular immunological recovery without GVHD is achieved within 6 months for most of the lymphocytes subsets excepted the CD4+ lymphocytes who need more than 12 months to recover. IgM and IgA recovery is also very slow.

Description: A monoclonal gammopathy of undetermined significance (MGUS) occurs in about 1% of the population over 50 years of age. Of these, about 20% evolves in Multiple Myeloma (MM); however, so far, predictive parameters of progression have not yet been identified.The aim of this study was to analyse the natural history of a cohort of non IgM MGUS and to identify whether or not there were laboratory parameters at diagnosis which can be utilized as prognostic markers of stable MGUS or progression to MM. From February 1974 to July 2001, 656 non IgM MGUS, whose clinical history was concluded (lost to follow up or died), have been followed at the Hematology of the University “La Sapienza” in Rome. The duration of follow up ranged from 2 months to 324 months, male/female ratio was 1.14, median age was 65 years (range 19–92). In each patient we evaluated: hemoglobin, platelet count, serum protein electrophoresis, serum concentration of monoclonal protein, serum calcium, creatinine, uric acid, BUN and percentage of bone marrow plasma cells.A monoclonal component (MC) of IgG type was documented in 543 patients (83%) while in 106 (16%) it was of IgA type, 6 patients had biclonal MC and 1 had a λ light chain MC; BJ proteinuria was detected in 78 (11%) patients at diagnosis. After a median follow up of 60.1 months (range 2–324) the MC remained stable in 496 patients (75%), whereas in 160 cases (25%) increased to evolve in MM. According to the literature, cumulative probability of progression to MM was 3%, 7% and 17% at 5, 10 and 15 years respectively. Differently from what observed by other investigators, in this cohort of pts, the MGUS of IgA type was not associated with a higher risk of progression to MM. The median time of progression to MM was 60.7 months (range 3–256) and factors associated with a more rapid progression to MM were advanced age and a higher number of bone marrow plasmacells. At diagnosis of MM, the concentration of the serum MC was significantly higher (P

Description: Background: The combination of thalidomide and dexamethasone is a rescue regimen for multiple myeloma (MM) in relapse, and for pretransplant in newly diagnosed patients. The bisphosphonate zoledronate mitigates bone resorption and possibly tumor growth and angiogenesis. The long-term utility of this combination in a newly diagnosed inner-city MM population with high prevalence of HIV-infection is addressed in this phase II trial. MM is reported with increased frequency in patients with HIV infection, and optimal treatment of MM in these patients is unknown. Methods: Of 30 consecutive enrollees, 22 (16F/6M) were evaluable. Mean age was 61 years (range = 43–82); all had skeletal lesions, and 27% (n = 6) were HIV+ (mean age = 47, range 46–50, all female). Patients received thalidomide 100 mg QD, dexamethasone 10–40 mg PO on Days 1–4, 9–12, and 17–20 monthly for six months, then on Days 1–4 monthly; and zoledronate 4 mg IV monthly, until progression or relapse. Baseline β2-microglobulin indicated high risk for all patients (mean = 6.2 μg/ml, SD = 3.8). At enrollment, 4 patients had AIDS, and 3 were on HAART. Results: Overall response rate (〉 50% decrease in M protein) was 72% (n = 13), and 83% (n = 5) in HIV+ patients. In 27% (n = 6), M protein levels were 〈 0.3 g/dL, including 3 HIV+ patients on HAART. In 23% (n = 5), M protein was reduced by 〈 50%. Median time to response was 2 months, and mean time on TDZ was 11 months. TDZ treatment decreased serum β2-microglobulin (P

Description: Purpose: Rituximab combined with chemotherapy has been recommended as first-line or second-line standard regimen in most subtypes of B-cell lymphoma in China by the 2004 National Comprehensive Cancer Network lymphoma therapy guideline. We have conducted a multicenter trial to evaluate the efficacy and safety of rituximab in combination with standard chemotherapy (CHOP) in patients with previously untreated or relapsed indolent and aggressive NHL. Methods: Patients received 4–8 cycles of rituximab plus CHOP every 21 days. For each cycle, rituximab (375mg/m2) was given on day 1 and CHOP started on day 3. CHOP consisted of cyclophosphamide 750mg/m2, doxorubicin 50mg/m2, and vincristine 1.4mg/m2 (maximum 2mg/dose) given intravenously on day 3, and oral prednisone 100mg on days 3–7. Results: A total of 221 patients were enrolled on the trial, 128 males and 93 females with a mean age of 49 years (range 10–83 years). The main lymphoma subtypes were small lymphocytic (15 patients, 7%), follicular (27 patients, 12%), and diffuse large B-cell (160 patients, 72%). In total, 56 patients had indolent NHL and 165 aggressive NHL. The overall response rate for all patients was 86% with 57% complete responses. In patients with indolent NHL the overall and complete response rates were 95% and 55% respectively. After a median 12 months follow up, progression-free survival in patients with indolent NHL was 88%±5% at 1 year and 83%±6% at 2 years. In the 160 patients with diffuse large B-cell lymphoma, the overall response rate was 88% with 61% complete responses, and after a mean follow-up of 6 months, predicted 1-year and 2-year progression-free survival were 88%±5% and 83%±7% respectively. Infusion-related adverse events occurred in 4% of patients, associated with the first infusion of rituximab. Subanalyses according to subtype, stage, IPI and other factors will be presented. Conclusion: Rituximab plus chemotherapy is an effective, well-tolerated treatment that achieves high response rates and long progression-free survival in both indolent and aggressive NHL.

Description: In this retrospective study we described the response and toxicity of a modified Magrath IVAC (mIVAC) regimen in 25 patients with refractory/relapsed aggressive non-Hodgkin lymphoma (NHL). The mIVAC consisted of ifosfamide 1,500mg/m2 (one-hour infusion beginning at 9:00; D1 to D5), mesna 300mg/m2 (bolus at hours 9:00, 13:00, 17:00; D1 to D5), citarabine 2,000 mg/m2 (two one-hour infusions beginning at 8:00 and 16:00; D1 and D2) and etoposide 60 mg/m2 (one-hour infusion beginning at 10:00; D1 to D5). Treatment was repeated every four weeks for a maximum of six cycles. Patients who achieved partial remission or complete remission after at least three courses were offered autologous stem cell transplantation (ASCT), if eligible. The median age was 37 years (range 18 to 59 years). Twenty-two (88%) patients had diffuse large B-cell lymphoma, fourteen (56%) had relapsed disease and 10 (40%) were considered high-intermediate and high risk by age-adjusted International Prognostic Index. The overall response rate was 68% (95% CI: 46%–90%). A total of 64 cycles were given, with a median of three courses per patient. Grade 3/4 neutropenia was observed after 85,6% of the courses, and grade 3/4 thrombocytopenia was observed after 87,5% of the courses. Grade 3/4 neutropenic fever occurred after 28% of the courses. Non-hematologic toxic effects were rare, predominantly grade 1/2. No toxic deaths were observed. Fifteen (88%) of the 17 responding patients underwent ASCT. With a median follow-up of 14 months, the median overall survival time for mIVAC sensitive patients was 16 months. This regimen may be feasible for patient with relapsed and refractory aggressive NHL in countries with inadequate numbers of hospital beds.

Description: Purpose: Rituximab is a chimeric anti-CD20 monoclonal antibody that was the first antibody approved by the FDA in the United States of America and SDA in China for the treatment of B-cell non-Hodgkin’s Lymphoma (NHL). It has shown significant efficacy and good tolerability in refractory and relapsed NHL. We have conducted a multicenter phase IV trial to evaluate the efficacy and safety of rituximab combined with standard CHOP chemotherapy in patients with newly diagnosed B-NHL. Methods: Patients with newly diagnosed, histologically proven CD20-positive NHL were eligible for the study. All patients received 4–6 infusions of rituximab (375mg/m2 per dose) in combination with CHOP chemotherapy, either concurrently (rituximab administered on the first day of each 21-day CHOP cycle) or sequentially (4–6 once-weekly infusions of rituximab followed by six 21-day cycles of CHOP). Each CHOP cycle consisted of cyclophosphamide 750 mg/m2, doxorubicin 50mg/m2, and vincristine 1.4mg/m2 (maximum 2.0mg/dose) given intravenously on day 1, and prednisone 100mg/day orally on days 1-5. Tumor responses were assessed at the end of treatment. Results: A total of 347 patients were recruited between February 2002 and December 2003. Of these 235 (68%) were male and 94 (27%) aged 〉60. The main lymphoma subtypes were diffuse large B-cell 196 (56%), follicular 41(12%), small lymphocytic/chronic lymphocytic leukemia 13(4%) and MALT 11(3%). Ann Arbor staging was as follows: stage I, 52 (15%); stage II, 80 (23%); stage III, 90(26%); stage IV, 105(30%); twenty patients (6%) could not be assessed. Of the 347 patients enrolled, 314 were evaluable for response. An objective response was observed in 94% of evaluable patients with a complete response (CR) in 56%, stable disease in 3.8% and progressive disease in 2.5%. The complete response rate was 63% for patients receiving 6 cycles of rituximab and 54% for those receiving four cycles of rituximab. No difference in response rate was observed between the sequential and concurrent groups. The most common adverse events were leucopenia in 122 patients (35%), nausea and vomiting 66 (19%), fever 39 (11%), rash 15 (4%) and asthma 4 (1%). Conclusion: The combination of rituximab and CHOP chemotherapy is an effective and well-tolerated treatment for patients with newly-diagnosed CD20-positive NHL. The safety and efficacy achieved in this study suggests that more than four doses of rituximab may be required for optimal efficacy.

Description: Thioguanine nucleotides (TGN) are considered the principal active metabolites exerting the antileukemic effects of mercaptopurine (MP). Numerous clinical studies have reported substantial inter-patient variability in intracellular TGN concentrations during continuation therapy of acute lymphoblastic leukemia (ALL). To identify genes whose expression is related to the intracellular accumulation of TGN in leukemia cells after in vivo treatment with MP alone (MP) or in combination with MTX (MP+MTX), we used oligonucleotide microarrays (Affymetrixâ HG-U95Av2) to analyze the expression of approximately 9,670 genes in bone marrow leukemic blasts obtained at diagnosis from 82 children with ALL. TGN levels were determined in bone marrow aspirates of these patients 20 hours after mercaptopurine infusion (1 g/m2 I.V). Because, as previously reported, patients treated with MP alone achieved higher levels of intracellular TGN compared to those treated with the combination, we used Spearman’s rank correlation to identify genes associated with TGN levels separately for the 33 patients treated with MP alone and the 49 with the combination (MP: median TGN: 2.46 pmol/5x106 cells, range: 0.01–19.98; and MTX+MP: median TGN: 0.55 pmol/5x106 cells, range: 0.005–3.31). Hierarchical clustering using these selected probe sets clearly separated the 33 patients treated with MP alone into two major groups according to TGN concentration (〈 2.46 and 〉 2.46 pmol/5x106 cells; n=60 genes) and two major branches were also found for patients treated with the combination (〈 0.55 and 〉 0.55 pmol/5x106 cells; n=75 genes). Interestingly, there was no overlap between the two sets of genes, indicating that different genes influence the accumulation of TGN when this drug is given alone or in combination with MTX. The association between gene expression profiles and TGN levels determined by leave-one-out cross-validation using support vector machine (SVM) based on Spearman correlation, was rho=0.60 (p

Description: Murine lymphoma models are commonly used to test the efficacy of idiotype-based vaccines for B-cell lymphoma prior to human trials. Mouse survival has been used as an indirect measure of the tumor response to the vaccine. In this study, we describe a multiparameter flow cytometric (MPFC) assay that enables direct assessment of the tumor at the inoculation site and detection of lymphoma metastases. In initial experiments, we show that 38C13 mouse lymphoma cells expressed an aberrant phenotype compared to normal splenocytes. The combination of CD45, CD19, B220 and an anti-idiotypic antibody (S1C5) along with a sequential gating technique defined the 38C13 cells with high sensitivity and specificity. The specificity of the MPFC assay for 38C13 lymphoma cells was greater than a gating strategy using light scatter and idiotype antibody binding. The MPFC assay was used to monitor 38C13 tumor kinetics in the C3H/HeN mouse and demonstrated tumor growth at the inoculation site and metastases to the lymph nodes and bone marrow. The presence of 38C13 metastases in lymphoid organs and bone marrow predicted by the MPFC assay were confirmed by histology and immunohistochemistry (IHC). The MPFC assay will enable investigators to monitor 38C13 lymphoma tumor response to individual vaccines or other lymphoma therapy prior to clinical trials. Furthermore, the MPFC concept could be readily applied to other models of human B cell lymphoma.

Description: CD123 constitutes the alpha subchain of the human interleukin 3 receptor (IL-3R). Interleukin 3 binds to its receptor with high and low affinities, induces tyrosine phosphorylation and promotes the proliferation and differentiation of haematopoietic stem cells. CD123 expression has been associated with a poor prognosis in AML and has been shown to be a useful marker in hairy cell leukaemia (HCL). However, it has been little studied in CLL, with data in the literature suggesting few cases of positivity. Here we studied CD123 expression by flow cytometry on fresh peripheral blood in 134 unselected, consecutive cases of CLL. We utilised anti CD123 PE (BD Pharminogen) in a dual combination with anti CD19 FITC (Coulter Immunotech) to target B cells. The results were correlated with expression of CD38 and ZAP70, markers of poor prognosis in CLL. CD123 was expressed in = 20% (median 33, range 20–92%) of cells in 32/134 cases (24%). Typical of CLL, the intensity of surface CD123 expression was weak-moderate, unlike HCL cases, where the intensity is moderate-strong. CD38 positivity was seen in 20/32 CD123+ cases (63%). Of the remaining 102 cases where CD123 was negative, a CD38 count was available in 92 cases and 29/92 (32%) were positive. ZAP70 positivity was seen in 18/32 CD123+ cases (56%). ZAP70 data was available in 98/102 CD123− cases and positivity was seen in 26/98 (27%). These findings represent the first account of a significant percentage of CLL cases expressing CD123 positivity. Furthermore we have been able to show that CD123 positivity strongly correlates with two known markers of poor prognosis ZAP70 (p= 0.004) and CD38 (p=0.004) (Chi-Square with Yate’s correction). CD123 may therefore be of prognostic significance in CLL and should be further investigated.

Description: 112 patients with senile acute myelocytic leukemia (AML), refractory or relapsed AML, and MDS-RAEBt entered this study to receive aclarubicin and low dose Arabinoside Cytosine (Ara-C) in combination with Granulocyte Colony Stimulating Factor (G-CSF) for evaluation of efficacy and tolerance of this CGA regimen. Low dose Ara-C was given at the dosage of 10mg/m2/12h, subcutaneously, D1-14 and aclarubicin 14mg/m2/d, intravenously, D1-4 (regimen A) or 7mg/m2 D1-8 (regimen B). Recombinant G-CSF was given at the dosage of 200μg/m2/d, subcutaneously, D1-14. We proved that overall response rate was 19/26 (73.1%) in senile AML, 48/62 (77.4%) in refractory AML, 12/18 (66.7%) in relapsed AML and 10/13 (76.9%) in MDS-RAEBt; and CR rate was at 8/26 (30.8%) in senile patients, 30/62 (48.4%) in refractory AML, 8/18 (44.4%) in relapsed AML and 5/13 (38.5%) in MDS-RAEBt, which were comparable in four groups of patients. 52 patients were followed up. Median progression free survival (PFS) and overall survival (OS) were 9.0±2.2 months and 11.0±1.6 months, respectively. The Kaplan-Meier estimated PFS and OS rate at 12 months were 40.73±8.15% and 42.85±8.23%, respectively. 1 year OS and PFS rate of the patients with CR were 60%±10.8% and 51.3%±13.7%, respectively, compared with the patients with PR, 17.7%±9.3% and 6.4%±6.1%, respectively. The toxic effects were very rare, mainly manifested as hematological changes, neutropenia and thrombocytopenia due to myelosuppression, which was around 70–80% exceeding NCI grade II. And non-hematological toxicities were not observed in this study. CAG regimen seems to be promising for the treatment of various categories of poor-prognosis AML or MDS-RAEBt, with acceptable toxicity.

Description: Recently we demonstrated that veto CTLs enhance engraftment of mismatched T cell depleted BM in recipient mice following reduced intensity conditioning. This desirable tolerance induction can be further enhanced by combining veto CTLs with CD4+CD25+ cells and Rapamycin. While these results are encouraging, they were largely based on models in which the resistant effector T cells mediating the allorejection are naive CTLp. However, considering that many patients undergoing BMT are presensitized by transfusions of different blood products, memory T cells could play an important role in graft rejection and, therefore, their sensitivity to veto cells could be critical to the implementation of the latter cells in BMT. Clearly, memory T cells respond under less stringent conditions to foreign antigens, compared to their naïve counterparts. In particular, they are programmed to be activated promptly, with a reduced requirement for costimulatory signals and therefore they might be more resistant to veto cells. To address this question we used the 2C mouse model, the CD8 T cells of which express a transgenic TCR against H-2d. The CD8 T cells bearing the TCR transgene can be followed by FACS using staining with a clontypic antibody (1B2) against the transgene. In this model, addition of veto CTLs was shown to inhibit expansion of CD8+1B2+ effector cells by induction of apoptosis which can be monitored by annexin V staining. Thus, in a total of 10 experiments the addition of 5% veto cells to 3 day MLR culture of naive 2C effector cells in the presence of H-2d stimulator cells, led to 76%±9% inhibition of expansion. In order to compare the sensitivity of memory cells in the same model, memory cells were established by immunizing 2C transgenic mice with 1x106 irradiated splenocytes from Balb/c donors (H-2d origin). Six weeks later, splenocytes were harvested and after Ficoll separation were shown to be enriched with memory CD8 T cells(CD44+high CD45Rb+ CD62L+, average in 16 different experiments was 73%±11). Upon addition of 5% veto cells to MLR culture of memory 2C spleen cells in the presence of stimulator cells, 78%±7% inhibition of 2C expansion was found. This veto activity was associated with increased apoptosis of allospecific memory CD8 T cells. Thus, in the absence of veto cells the CD8+1B2+ memory cells exhibited a low level of Annexin V (6%±3%) while in the presence of 5% veto cells, a high level of Annexin V (25%±9%) was detected. The deletion of the 2C memory effectors, as previously shown for naive 2C cells, is largely dependent on the presence of Fas-FasL interaction, as indicated by using memory cells from 2C- lpr mice that lack Fas receptor on the cell surface. Upon addition of veto cells to MLR culture with 2C memory spleen cells from lpr mice, only a minor reduction of expansion (5.5%±6% in the presence of 10% veto CTLs) was detected. In conclusion, these results suggest that veto cells can delete memory effector cells as efficiently as exhibited on naive effector cells and by a similar Fas-FasL dependent mechanism. This finding might have significant implications not only for BMT, but also for the treatment of autoimmune diseases in which memory T cells play a major role.

Description: The Jak-STAT pathway is responsible for signal transduction by a large number of cytokine receptors. While this pathway is normally tightly regulated, constitutive STAT3 and STAT5 activation is frequent in hematologic malignancies and contributes to oncogenesis. We took advantage of the IL-3-dependent pro-B Ba/F3 cell line to analyze the mechanisms of constitutive STAT activation in vitro. Ba/F3 cells transfected with a mutated hIL-9R (Ba/F3 Phe116) poorly respond to IL-9. However, after selection with this cytokine, we obtained cell lines that proliferated well in IL-9. After cytokine withdrawal, those cells, in contrast to the parental cells, gave rise to autonomous Ba/F3 cell lines showing constitutive STAT5 activation, and that were highly tumorigenic in vivo. This process mimics the multistep process of oncogenesis occurring in vivo. To dissect the mechanisms involved in this cellular transformation, we analyzed, using cDNA microarrays, genes expressed in one of these autonomous lines. Jak1, the Jak kinase associated to the IL-9 receptor, was found to be upregulated compared to parental cells. It was the key genetic event involved in this transformation since ectopic Jak1 overexpression increased the sensitivity to IL-9 and allowed, after a second selection step, for spontaneous progression towards autonomous cell lines with constitutive STAT activation. Using Jak1 mutants, we showed that the generation of autonomous lines was dependent on the tyrosine kinase activity of Jak1 and on a functional FERM domain, the domain that mediates association with the receptor. These results were extended to the other Jak family members. Therefore, we propose that Jak overexpression can be considered as one of the oncogenic events leading to the selection of cells highly responsive to growth factors and, after a second selection step, to autonomous cells with constitutive activation of the Jak-STAT pathway. This process could be extended to human malignancies and might explain, for instance, the constitutive STAT6 activation in primary mediastinal large B cell lymphomas where Jak2 overexpression was described.

Description: In 1993, we initiated an international trial in newly diagnosed adult (age 15–60 years) ALL patients. 1637 patients have been entered and data analyzed as of June 2004. All patients receive two induction phases of therapy and CNS prophylaxis (or therapy if CNS involved) as assessed by lumbar puncture for cytospin. Patients age ≤ 50 yr in CR1 who have a matched-related donor (MRD) undergo an allograft, and the remainder in CR1 are randomized either to an autograft or 4 cycles intensive consolidation and maintenance chemotherapy. Matched unrelated donor (MUD) allograft is an option in Philadelphia chromosome (Ph) positive patients. 46 (3%) patients had CNS leukemia at presentation. Median (range) age was 29 (16–56) years, and 5 were Ph+. Five patients never attained CR1 (3 died early during induction on days 4, 25 and 36). 41/46 (89%) patients attained CR1. 23 patients underwent transplant, 17 in CR1 (10 MRD, 2 MUD, and 5 autografts), while 3 MUD and 3 MRD allografts were performed for 〉 CR1. 31/46 patients died, 12 of leukemia, 8 of infection, 3 of GVHD and 8 other causes. 12 patients died in remission due to infection (5), bleeding (2), GVHD (3), and visceral organ failure (2). 15 patients remain alive, one Ph+ in relapse at 58 mo and 14 (30%) Ph- remain in continuous CR at a median 67 (range: 2–133) mo after diagnosis. Treatments for these 15 long-term survivors were: MRD allograft (6 @ 45,53,63,67,98,120 mo), MUD allograft (1 @ 67 mo; performed in relapse), chemotherapy only (6 @ 2,55,87,98,125,133 mo) and autograft (1 @ 31 mo). In this study we noted a low incidence of CNS leukemia but we cannot exclude avoidance of enrollment due to suspected CNS involvement. Adult Ph- ALL patients with CNS leukemia at diagnosis can attain long-term disease-free survivorship using transplantation as well as conventional chemotherapy. Therapy (and status) No. Pts Survivors in CR (mo) Induction death 3 Died: no remission (resistant disease) 2 MUD without remission 1 67 MUD in CR1 2 MUD post-relapse 2 MRD in CR1 10 45,53,63,67,98,120 MRD 〉CR1 3 58 (in relapse) Auto (CR1) 5 31 Chemotherapy (CR1) 18 2,55,87,98,125,133 Total 46

Description: STI571 is a highly effective drug for the therapy of CML, but there is still drug resistance, especially in blast crisis. To study the possible mechanisms of resistance to STI571, we established the BCR/ABL+ cell line with resistance to STI571 (K562-R) in vitro by culturing a wild-type K562 cells (K562-W) in gradually increased concentrations of STI571 over a period of months. Trypan blue staining, MTT assay and Hoechst 33342 staining confirmed that K562-R can live steadily at 0.5umol/L STI571. Furthermore MDR-1 expression assay, sequence analysis, fluorescence in situ hybridization(FISH) and cDNA array were used to study the potential mechanisms of acquired resistance. The MDR-1 expression percentages of K562-W and K562-R with FASC analysis were 2.68% and 1.39% respectively. No point mutant in the BCR/ABL ATP-binding site was detected and the copies of BCR/ABL fusion gene were found increased in K562-R by FISH analysis. By a expression profile of cDNA microarray, 327 genes’ expression were found down-regulating including one of homo sapiens protein tyrosine phosphatase genes(PTPRF) and 335 genes up-regulating including homo sapiens hematopoietic cell-specific Lyn substrate 1 gene(HCLS1). Our studies proved the possible mechanism of K562-R resistance involved amplification of BCR/ABL fusion gene and increase of phosphorylation activity in this cell line.

Description: Background: The breakpoints in the cytogenetic lesion in Burkitts lymphoma are very far apart such that the t(8,14)9q24;q32) is not amenable to standard polymerase chain reaction analysis. Long range polymerase chain reaction (LD-PCR) with its enzyme mix, appears to be capable of amplifying the t (8;14)(q24;32) in the majority of published sporadic Burkitts lymphoma analyses. The utility of t(8;14)(q24;q32) LD-PCR for routine use in the diagnosis of African and AIDS-related Burkitts lymphoma has not been studied. This study aims to analyze bone marrow of known African and AIDS-related Burkitts Lymphomas using LD-PCR and to establish if this procedure is suitable for routine diagnostic use. Materials and methods: High molecular weight DNA was extracted from stored unstained bone marrow slides of previously diagnosed Burkitts lymphoma. Three hundred nanograms of patient and control DNA were amplified as per published LD-PCR methods. Each DNA sample was amplified with up to three pairs of MYC/IgH primer sets. The resulting amplicons were separated on a 0.8% agarose gel and their size compared to that of positive t(8;14)(q24;q32) controls, tPa controls and molecular weight markers. Results: DNA was extracted from 74 Burkitts lymphoma bone marrow slides. Only 41 of these were amplifiable with the tPa primers and therefore suitable for further analysis. A t(8;14)(q24;q32) specific product was demonstrable in only 6 of 41 patients. Conclusion: In this t(8;14)(q24;32) LD-PCR analysis of known Burkitts lymphomas comprising largely of African and AIDS-related variants, the procedure appears to be labour intensive and costly with a low diagnostic yield. These results may reflect the fundamental molecular differences between the sporadic and African or AIDS-related Burkitts lymphomas.

Description: Activated protein C (APC) resistance is the most frequent hereditary defect associated with deep vein thrombosis (DVT). Over 80% of the APC resistance phenotypes can be explained by the Factor V Leiden (FVL) mutation. This defect is caused by a point mutation in the factor V gene resulting in a replacement of the amino acid Arg 506 by a Gln residue. For patients who are at risk of thrombosis or who have active thrombosis, it is of interest to be able to screen for the FVL mutation in a easy and reliable manner. There are two types of assays for the detection of FVL. The genotype is reliably detected by PCR technology, but this technology is not readily available to all laboratories nor is it inexpensive. The phenotypic expressions of the defect can be identified by plasma-based functional assays, of which several are commercially available. The functional Pefakit® APC-R FVL assay (Pentapharm; Basel, Switzerland) (APC-R) is a new plasma-based clotting assay developed to overcome the limitations of the current assays, including sensitivity and specificity. Additionally, the APC-R assay is more informative as results specify heterozygous / homozygous opposed to a report of normal / abnormal by all other assays. The APC-R assay is designed to specifically act at the level of the prothrombinase complex using a factor V dependent prothrombin activator isolated from the snake venom of the Notechis scutatus. We investigated the phenotypic and genotypic expressions of FVL in patients with cancer and acute symptomatic DVT and/or PE (n=67). Normal healthy volunteers (n=50) collected in-house served as controls. DNA isolated from buffy coats obtained from citrated anticoagulated blood was used to profile FVL using standard PCR probes. Citrated plasma was used to determine APC-R by the functional Pefakit® assay. Of the 67 patients profiled by molecular analysis, 5 (7%) were heterozygous and 1 (1%) was homozygous for the FVL defect. In the normal healthy volunteers, 3 (6%) were heterozygous and 1 (2%) was homozygous for FVL. Using the APC-R functional plasma-based assay, 4 cancer patients (6%) were heterozygous and 2 (3%) were homozygous. In the normal volunteers, 4 (8%) were heterozygous and 1 (2%). was homozygous with the APC-R assay. The APC-R functional plasma-based assay did not miss any FVL patient shown positive by PCR. The heterozygous cancer patients detected with the functional assay were detected as heterozygous with the PRC method; one patient detected as homozygous with the functional method was determined to be heterozygous by PCR. In the normal healthy volunteers, 1 volunteer that was heterozygous by the plasma based assay was negative by PCR. This study demonstrates that the prevalence of the FVL defect in cancer patients with thrombosis is similar to the normal healthy population. Although additional studies to validate the sensitivity/specificity of the APC-R assay and to establish its positive / negative predictive values are needed, the results of this first investigation suggest that the APC-R assay may provide more useful results than other commercially available assays for the detection of FVL. The results of this study showed a good correlation of the functional FVL. APC-R assay with the reference standard molecular PCR method. These data suggest a practical role of this new assay in the clinical laboratory diagnosis of FVL.

Description: In children with acute lymphoblastic leukemia (ALL), failure due to therapy-related myeloid leukemia (t-ML) is a devastating complication. Using a target gene approach, only a few host genetic risk factors for t-ML have been defined. Microarray analysis of gene expression allows for a more genome-wide approach to identify possible genetic risk factors for t-ML. We assessed gene expression profiles (12625 gene probe sets) using oligonucleotide-based arrays in diagnostic ALL blasts from 228 children treated on St. Jude ALL protocols (Total XIII) that included etoposide; 13 of these children developed t-ML. A group of 83 probe sets were significantly related to the time-dependent risk of t-ML, with principal component analysis plot (right panel) separating patients who developed t-ML from the others. Hierarchical clustering of the 83 probe sets grouped patients into 3 clusters (n=163, n=52, n=13), with the cumulative incidence of t-ML being significantly higher in the last cluster (p 〈 0.0001, left panel) compared to those of the other gene-expression-defined clusters. Figure Figure A permutation test indicated that probe sets selected by chance are unlikely to obtain the observed distinct clusters (p=0.045). Distinguishing genes included transcription-related oncogenes (v-Myb, Pax-5), cyclins (CCNG1, CCNG2 and CCND1) and Histone H4. Common transcription factor recognition elements among similarly up- or down-regulated genes included several involved in hematopoietic differentiation or leukemogenesis (Maz, PU.1, FOXO4). This approach has identified several genes whose expression differentiates patients at risk of t-ML, and provides targets for assessing the germline predisposition to leukemogenesis.

Description: Bernd Engelmann Vascular Biology and Hemostasis, Inst. of Clinical Chemistry, Ludwig-Maximilians-Universität, Munich, Germany Following addition of fibrillar collagen to whole blood in order to mimick the starting process of hemostasis, we unexpectedly observed that tissue factor (TF), the central initiator of coagulation, was exposed within 3–5 min in association with CD15 and CD14 positive blood cells. A series of experiments revealed that the TF presentation was restricted to conjugates of neutrophils (and monocytes) with platelets. To verify the source of the TF, isolated neutrophils and platelets were evaluated for the presence of TF. Using a double sandwich Elisa, the washed platelets were found to contain TF. Conversely, TF was undetectable in the neutrophils. When searching for the intraplatelet location of TF by immunoelectron microscopy (IEM), TF was observed to reside in the alpha-granules and in the surface connected system. No TF was present in the cytoplasma and the dense granules. In response to activation, platelet TF was translocated to the cell surface by fusion of the alpha-granules with the plasma membrane. The externalized TF was found to cluster on platelet filopodia. Inspection of rapidly isolated buffy coat preparations confirmed the absence of TF from the neutrophils. Stimulation of TF-dependent factor Xa formation by the activated platelets was markedly amplified by the isolated neutrophils. This required neutrophil-platelet conjugate formation, as evident from inhibition by antibodies targeting PSGL-1 and CD18. To assess whether the TF triggered coagulation was connected to the platelet recruitment, we evaluated the participation of the ADP system. Disrupting the interaction of ADP with its platelet receptors P2Y12 and P2Y1 suppressed the TF activity in the neutrophil-platelet conjugates. Since the TF exposing filopodia represent preferential sites for the formation of microparticles (MP), we isolated the total pool of circulating MP from whole blood, known to be mainly derived from the platelets. Then, the MP were separated by cell sorting. In MP positive for the platelet specific CD42b, TF could be detected and quantified by western blotting and Elisa. Moderate increases in MP number excessively stimulated blood based TF activity in the presence of platelets and in whole blood. Since activated platelets are known to secrete tissue factor pathway inhibitor (TFPI), an anti-TFPI antibody targeting the Kunitz-2 domain of TFPI was included into the suspensions of the activated platelets. Thereby the TF activity of the isolated platelets was enforced, while the activity in the presence of the neutrophils remained unaffected, suggesting that TFPI partially masks the functional competence of the platelet TF. The potential contribution of platelet-collagen interactions for the activation of coagulation in vivo was analyzed by injecting collagen into the venous blood of mice. Local fibrin formation was documented in pulmonary vessels by EM, and systemic thrombin generation was revealed by increased thrombin-antithrombin complexes. In mice deficient for the P2Y1 ADP receptor, the thrombin generation was markedly reduced, indicating a basic role for the platelet-triggered coagulation during thrombus growth. In conclusion, the intravascular tissue factor enables the entire coagulation system to proceed on the plasma membrane of a single blood component, the surface of the activated platelets. Consequently the coagulation start can be regulated within the platelet aggregate, allowing fibrin formation to be flexibly adjusted to the size of the thrombus and the duration of its development.

Description: Derangement of cellular immunity is central in the pathophysiology of adult autoimmune/idiopathic thrombocytopenic purpura (ITP). Herein we investigated cytokine gene expression in peripheral blood mononuclear cells (PBMCs) of adult chronic ITP patients and attempted to correlate cytokine polarization with the degree of thrombocytopenia. We used semiquantitative reverse-transcriptase–polymerase chain reaction (RT-PCR) to measure the expression of type-1 (interleukin-2 [IL-2], interferon γ [IFN-γ]) and type-2 (IL-4, IL-5, IL-10, IL-3, IL-13) cytokines by PBMCs from 21 patients and 11 controls. Plasma transforming growth factor β1 (TGF-β1) levels were measured by enzyme-linked immunoassay (ELISA). T helper 1 (Th1)/Th2 ([IL-2 + IFN-γ]/[IL-4 + IL-5]) cytokine mRNA ratios, thought to reflect the Th deviation of the pathogenic disease-specific T cells, and type-1/type-2 mRNA ratios, thought to reflect the overall immune response polarization, were significantly increased in ITP patients. The Th1/Th2 ratio was inversely correlated with platelet counts. TGF-β1 levels appeared suppressed in patients with active disease, though not significantly. Our findings show a clear type-1 cytokine polarization of the autoimmune response in adult ITP that persists irrespective of disease status.

Description: Polycythemia vera (PV) is a myeloproliferative disease characterized by accumulation of erythrocytes and cells of the myeloid and megakaryocyte lineages. Although genes like PRV-1 and PTP-MEG2 have been implicated in the pathology of PV, there is no consensus on their importance in the disease process. Progenitor cells from PV patients can grow in the absence of erythropoietin, and are hypersensitive to a variety of other growth factors. This suggests that polycythemic hematopoietic progenitor cells possess a significantly different genetic program. We tested this idea by molecular profiling hematopoietic progenitor cells (CD34+) from PV specimens and normal donors. We purified CD34+ cells from the marrow of 10 PV patients and harvested total RNA. Biotinylated cRNA was made through two rounds of linear amplification, and hybridized to Affymetrix HGU133A genechips. CD34+ cells from marrow mononuclear cells of 5 normal controls were processed similarly. The resulting datasets were normalized to the median across chips and across genes. Unsupervised hierarchical clustering showed that PV samples had a distinct gene expression profile from the controls. We then performed supervised clustering using a non-parametric t-test (Wilcoxon rank sum test) using the Benjamini and Hochberg multiple testing correction held to a p-value of 0.01 to determine genes that were significantly different between disease and normal samples. Using these stringent criteria, there were 331 genes that reached significance. Strikingly most of these were decreased in expression compared with control CD34+ cells and only 34 genes were upregulated in PV. A 35 gene predictor set was discovered through the use of a k-nearest neighbor metric. This set was 100% accurate for the prediction of PV in a leave one out cross-validation approach. Among these genes are EVI1, a known oncogene and one of only two genes upregulated in PV on this list, and the putative tumor suppressor genes TUSC4 (NPR2), NDRG1 and KLF4. Also among the predictor genes is BAALC, a gene expressed in normal CD34+ cells and known to be a prognostic indicator gene for acute myeloid leukemia.

Description: Recent studies using direct stimulation of PBMC from CMV-positive individuals with optimal CTL epitope peptides have identified new antigens that are targets of the host immune response. The growing number of targets of the cellular immune response has prompted an evaluation of which antigens are potentially associated with protective immunity. A panel of seven human cytomegalovirus (CMV) epitope peptides and the corresponding MHC-I tetramers was used to evaluate cellular immunity in six healthy seropositive donors and in six hematopoietic stem cell transplant recipients (HSCT). Broad CMV-specific responses were found to epitopes within several CMV polypeptides that were restricted by multiple HLA alleles in several individuals. The results document the simultaneous expansion of several of these CD8+ T-lymphocyte populations following allogeneic HSCT. The combined levels of CMV epitope-specific T-cells exceeded 20% of CD8+ T-lymphocytes in some individuals. The cytotoxic functionality of 26 different populations of CMV-specific T-lymphocytes detected within this group of 12 individuals was addressed by utilizing a recently described assay that measures transient surface levels of the lysosomal membrane proteins LAMP-1 (CD107a) and LAMP 2 (CD107b) after peptide stimulation. This degranulation/mobilization assay can be combined with tetramer staining of antigen-specific CD8+ T-lymphocytes, and has potential as a surrogate marker for cytotoxic function. We found that a significant proportion of CD8+ T-lymphocytes specific for epitopes within the CMV pp65 and pp50 gene products had functional potential as measured by this assay (median percentage of cells within 14 T-cell populations staining with pp65 or pp50 tetramers that degranulated on stimulation with cognate peptide = 26.0% and 19.8%). By contrast, CD8+ T-lymphocytes specific for epitopes within the CMV IE-1 gene product had markedly reduced functionality (median percentage of cells within 12 T-cell populations staining with IE-1 tetramers that degranulated on stimulation with cognate peptide = 5.6%). This difference was significant (p= 0.003 by an F-test after adjusting for HLA and using interaction of subjects and epitopes as the error). This reduced degranulation efficiency of IE-1-specific T cells is consistent with their inefficient cytotoxic recognition of CMV-infected autologous fibroblasts. Further characterization of this functional dichotomy includes comparison of cell surface marker phenotype and cytokine release in response to antigenic presentation. These functional differences between T-lymphocyte populations within the same individual have implications for choosing antigens that are both protective and necessary to include in a CMV vaccine for transplant patients at risk for infectious complications after viral reactivation.

Description: The prognosis for patients with mantle cell lymphoma (MCL) treated with conventional chemotherapy remains poor. Dose escalation and stem cell transplantation has been increasingly employed in an attempt to improve the outcome in these patients. However, due to the advanced age of many patients with MCL, high dose therapy and allogeneic stem cell transplantation is particularly hazardous. Reduced intensity allogeneic transplantation (RIT) may reduce the toxicity of allogeneic stem cell transplantation, facilitate allogeneic engraftment and graft versus lymphoma reactions. However, the results reported to date with this treatment modality have been based on small numbers of patients and provide conflicting results. We have therefore analysed the outcome of a large cohort of patients with MCL reported to the EBMT registry who have undergone RIT. A total of 144 patients (123 male) with a histological diagnosis of MCL were reported by 81 centres. The median age at transplant was 49 years (range 28–68 years) and the median time from diagnosis to transplant was 25 months (range 0.25–13.2 years). The patients had received a median of 2 (range 1–5) lines of prior chemotherapy and 60 (42%) had undergone a prior high dose procedure. At the time of RIT 100 patients had chemosensitive disease, 22 chemoresistant disease and 22 had untested relapse. Patients underwent conditioning with reduced intensity regimens prior to transplantation with allogeneic peripheral blood stem cells (122), bone marrow (20) or both (1). Fully matched sibling donors were used in 109 cases, matched unrelated donors in 21 and 9 patients received mismatched stem cells. 123 of 126 patients assessable for engraftment demonstrated sustained engraftment. With a median follow up of 9 months 84 patients remain alive and 60 have died (15 from progressive disease and 45 from non-relapse mortality). The transplant related mortality (TRM) was 12% at 100 days but by Kaplan-Mier analysis the TRM was 35% at 1 year and 50% at two years. In univariate analysis there was a non-significant trend to a higher TRM in patients with chemoresistant disease (p=0.067) and those with a prior transplant (p=0.062). Patient age and the number of lines of prior therapy had no impact on TRM. At two years following transplant 57% of patients had evidence of disease relapse or progression which was significantly worse in those with chemoresistant disease prior to transplant (p=0.02). The overall survival (OS) at 1 year and 2 years was 55% and 31% respectively and was worse for patients with chemoresistant disease. The progression free survival (PFS) at 1 and 2 year was 43% and 26% respectively. Only disease status at transplantation predicted for a worse PFS. Acute GVHD (grade II-IV) developed in 52 patients and chronic GVHD in 23 patients. Although the early transplant related toxicity is low there remains a significant TRM following RIT for MCL and consequently a low progression free survival. Patients with chemoresistant disease have a particularly poor outcome.

Description: Oral anticoagulant treatment with vitamin K antagonists has been encumbered by the need for interval monitoring of INRs to adjust dosage. Point of Care (POC) testing of capillary blood has come into widespread use because of the advantages of rapid analysis and dose adjustment. To assess the reliability of this procedure, we compared INR results from 2 POC devices from different manufacturers - POC1 and POC2 - and compared these results to standard assays on venous samples with a BCS automated analyzer. 78 patients on warfarin for at least 3 months were studied. Results of 56 patients with POC2 and 76 patients with POC1 were compared to the BCS. Capillary blood samples were taken within 30 minutes of the venous draw. The therapeutic INR range was considered to be 2.0–3.5. The bias plots for the 2 POC methods compared to the reference INR are shown in the 2 Figs, below. Within the therapeutic range, 71% and 85% of the results correlated with BCS for POC1 and the POC2, respectively. Both instruments showed the greatest variation from the reference measurement at INR values greater than 2.5 Both POC methods demonstrated clinically relevant overestimates of INR(see table). Figure Figure Figure Figure Agreement % of Reference INR Values to POC INR Reference INR* POC1-subtherapeutic POC1-therapeutic POC1-supratherapeutic POC2-subtherapeutic POC2-therapeutic * INR = 2.0–3.5 BCS-subtherapeutic 77% 23% - 63% 37% BCS-therapeutic 4% 71% 25% 5% 85% BCS-supratherapeutic - - 100% - 33% Conclusion: This study raises a significant question rearding the accuracy of POC methods for INR determinations. Despite ease of use and immediacy of results, the lack of correlation, even under study conditions, and the potentials for under-and overdosing makes relying on these devices problematic.

Description: In the previous ASH Meetings, we have reported that thrombin/antithrombin complex in association with high density lipoprotein (TAT/HDL) directly stimulated proplatelet formation (PPF) of human or murine megakaryocytes. In this study, we investigated the signal transduction pathways responsible for platelet production from megakaryocytes using human megakaryoblastic cell line (MEG). TAT/HDL stimulated MEGs to induce the many platelet-like particles (PP) around the cells in the serum free culture during 12~24 hrs (Fig. 1). The frequency of MEGs with PP increased with the increase of TAT/HDL in a dose dependent manner (0 μg/ml: 1.8±0.5%, 50 μg/ml: 30.8±4.5%, 100 μg/ml: 46.5±7.8%, 200 μg/ml: 57.8±8.3%, mean±SD, n=4). The frequency of MEGs with PP, expressed as a pecentage of the control, was significantly decreased after the incubation with TAT/HDL(150 μg/ml) and Y27632, a specific inhibitor for Rho kinase(ROCK), (Y27632 10 μM: 61.3±7.9% of the control (p

Description: AMN107 is a novel aminopyrimidine ATP-competitive inhibitor of Bcr-Abl. In proliferation assays AMN107 ≥10-fold more is potent than IM against Bcr-Abl expressing cell lines. AMN107 is effective against cell lines expressing the following IM-resistant Bcr-Abl mutants: Glu255Val, Phe317Leu, and Met351Thr. In addition, preliminary studies indicate that AMN107 has similar potency to IM against c-Kit and PDGFR-dependent cell proliferation. The ↑ potency and broader spectrum of activity of AMN107 against Bcr-Abl, relative to IM, may result in clinical benefit for pts with CML or Ph+ ALL. In the phase I portion of this phase I/II study, pts with IM-resistant CML-AP, CML-BC, and Ph+ ALL were eligible for treatment with AMN107 as an oral daily dose. 21 pts [median age:61 yrs (range 29–77); 15 male 6 female; performance status: 0(12 pts), 1(6 pts) or 2(3 pts)] have been enrolled in the following dose cohorts (mg/day): 50(7 pts), 100(7 pts), 200(7 pts) and have been on treatment for 8–70 days. Disease types: CML-AP (12 pts), CML-BC (6 pts), Ph+ ALL (3 pts). Intra-pts dose escalations were permitted for persistent disease in peripheral blood and/or marrow. 6/7 pts in the 50mg dose level, and 7/7 pts in the 100mg dose level have dose-escalated. No AMN107-related AE have been observed. Biologic activity, defined as at least a 50% ↓ in blasts or basophils in the peripheral blood and/or marrow lasting for at least 7 days was observed in 0/7 pts treated with 50mg/day, 4/13 pts treated with 100mg/day, and 7/15 pts treated with 200mg/day. Of the 7 pts who demonstrated biologic activity at 200mg/day, 2 pts with CML-AP had complete hematologic responses in marrow, and 1 pts with CML-AP had return to CP in marrow. Pts were not pre-selected for treatment based on mutational status of Bcr-Abl. Mutational analysis is being performed on all pts and will be correlated with response. Initial data reveal Bcr-Abl mutations in the majority (〉 80%) of baseline pts samples. Preliminary data from the 50mg cohort comparing baseline peripheral blood samples with day 2 samples showed: significant ↑ in apoptosis as determined by mitochondrial potential, reduction in proliferation of CD34+ cells as measured by BrdU incorporation, and significant reductions in STAT1 phosphorylation. Reduction in CRKL phosphorylation in CD34+ cells was observed. Similar changes were noted in marrow. PK samples were collected on days 1, 2, 8, 15, 22 & 28 of cycle 1 and at time of any intra-pts dose escalation. Pharmacokinetics after 1 daily oral dose of AMN107 were characterized as little accumulation after multiple administrations with moderate inter-pts variability in exposure. Peak concentrations of AMN107 were generally achieved by 3 hrs post-dose. Preliminary PK data support 1-daily dosing. This phase I study, AMN107 given orally appears to be well tolerated with biologic effects in some pts treated with ≥100mg/day & marrow responses in some pts treated with 200mg/day. Once MTD has been determined, pts will be enrolled to multiple expansion cohorts in the phase II portion to assess activity.

Description: Background: Previous research has shown early hemoglobin (Hb) response to epoetin alfa (EPO) therapy is associated with reduced transfusion requirements, higher Hb response rates, quality of life score improvements, and decreased EPO drug utilization. This subgroup analysis of elderly (age 〉/=65) patients (pts) with chemotherapy-related anemia (CRA) assesses the benefit of an early Hb response in this distinct population. Methods: Data from three large multicenter EPO clinical trials were evaluated. In EPO 1, pts received 10,000 Units TIW with potential escalation to 20,000 Units TIW. In EPO 2 and EPO 3, pts received 40,000 Units QW with escalation to 60,000 Units QW. Pts eligible for this analysis were 〉/=65 years of age with a non-myeloid malignancy, had a baseline Hb /=1g/dL Hb rise following four weeks of EPO therapy, independent of transfusion in the prior 28 days. Three outcomes of pts who exhibited early Hb response were compared to those of pts who did not: proportion of pts requiring transfusion, subsequent Hb response (Hb rise 〉/=2 g/dL independent of transfusion), and average weekly EPO dose. Results: Early Hb response was observed in 54.1%, 47.5%, and 47.2% of pts from EPO 1, 2 and 3, respectively. In all trials, early responders had markedly lower transfusion use (EPO 1: 7.6% v 22.5%, p

Description: Several examples suggest a relationship between in vitro migratory capacity and bone marrow (BM) homing. Pertussis toxin (PTX) is a potent inhibitor of Gi protein coupled receptor signaling and as such, it blocks hemopoietic progenitor cell (HPC) migration in vitro. However, contrary to expectation, no effects on BM homing were observed in previous studies. We therefore re-examined the effect of PTX on BM homing of HPC. We found that prolonged incubation of BM cells or fetal liver cells with PTX inhibited their BM homing in irradiated or non-irradiated recipients by 〉75%. A concomitant increase in circulating CFU-C in blood provided added assurance that the data were not due to non-specific toxicity. This inhibition of BM homing was of functional consequence, since PTX-treated cells provided impaired radioprotection and markedly decreased short-term engraftment. Detailed studies showed that inhibition of in vitro migration and ribosylation of Gi proteins were complete only after extended incubation with PTX, whereas short-term incubation for 1–2 h, as used in previous studies, was insufficient. In addition, the incubation of BM cells with SCF may have exaggerated the negative effect of PTX on the inhibition on BM homing. We next sought to test the basis of the residual 25% homing of PTX treated cells. As PTX did not directly inhibit hemopoietic cell adhesion, we tested homing of PTX-incubated and control BM cells in genetic models deficient in certain adhesion molecules on either the hemopoietic cells or the hemopoietic microenvironment. BM homing of PTX-treated α4integrin-/- BM cells transplanted into wild-type (WT) mice, or PTX-treated WT cells transplanted into EPselectin-/- recipients inhibited BM homing significantly stronger than either modality alone, to 〉90% and 〉95%, respectively. Of note, untreated WT cells have normal homing in EPselectin-/- mice. These data demonstrate cooperativity between Gi blockade and α4integrin or endothelial selectin blockade in inhibiting BM homing of progenitor cells. Absence of β2integrin or L-selectin in the presence or absence of PTX did not inhibit BM homing. In summary, these studies show that Gi protein signals are required for BM homing of progenitor cells. PTX is a very strong inhibitor of BM homing, suggesting that it may inhibit convergent signals from more than one mediator. We further show that the residual BM homing of migration-impaired BM cells is due to participation of α4 integrin on the transplanted cells, or to endothelial selectins on the host side.

Description: Short-term administration of rhG-CSF is a safe and effective procedure for mobilization of allogeneic PBPC in healthy related and unrelated donors. Mild to moderate bone pain is the main side effect occurring in about 80 % of donors, whereas major complications are extremely rare. Recently we observed a patient, who developed life-threatening autoimmune hyperthyreoidism two weeks after rhG-CSF stimulated unrelated PBPC donation (Kroschinsky et al., Haematologica 2004). Anecdotal reports in the literature describe thyroid dysfunction in cancer patients who received rhG-CSF or rhGM-CSF to enhance neutrophil recovery after chemotherapy. In patients with autoimmune disorders who had been treated with rhG-CSG for secondary neutropenia a flare up of disease specific symptoms was observed. Although the current evidence is poor, there might be a potential role of rhG-CSF in thyroid dysfunction and autoimmunity due to its pleotropic effects on different types of cellular and humoral immune effectors. Therefore we prospectively investigated 104 (27 female, 77 male, median age 36 yrs) unrelated PBPC donors for organ and tissue specific autoantibodies. Mobilization treatment consisted of 7.5 μg/kg body weight lenograstim (Granocyte™, Chugai Pharma Inc., Tokyo, Japan) for five days. Leukaphereses were performed on day 5 and 6. Blood samples were taken at pre-donation health-check (before rhG-CSF stimulation, sample 1), at day of apheresis (sample 2) and four weeks after donation (sample 3). The panel of 21 autoantibodies which was tested sequentially is listed in the table. The frequency of positive findings varied considerably between the different antibody specificities. Whereas none of the donors showed reactivity for antibodies to LKM-1, insulin or proteinase-3, antibodies to endothelial antigens were detected before, at and after donation in 16.5, 13.4 and 17.9 % of donors, respectively. No significant differences were found comparing the number of positive results of the sequential antibody testing. However, there was a significant increase in antibody concentrations measured by immunoassays from sample 1 to sample 3 for anti-TSHR (p=0.02), anti-PR3 (p

Description: The adult bone marrow produces 2x1011 red blood cells daily to maintain the oxygen carrying capacity of peripheral blood. Certain pathologic conditions such as blood loss or hemolysis that result in increased oxygen demands lead to a physiologic bone marrow response characterized by increased rate of erythropoiesis and expansion of bone marrow proerythroblasts, the precursor cells that differentiate into mature red blood cells. We studied the mechanisms by which erythropoietin (EPO) and stem cell factor (SCF) regulate the expansion of primary human proerythroblasts. Using liquid cultures of peripheral blood CD34+ cells isolated from healthy volunteers, we generated uniform populations of transferrin receptor (CD71)+ human proerythroblasts. We established a serum-free culture model to study the effect of EPO and SCF on the survival and proliferative capacity of the cells. Primary proerythroblasts failed to survive in the presence of EPO or SCF alone, but exhibited marked synergistic proliferation in response to EPO plus SCF, exhibiting one log expansion in 5 days under serum-free conditions. Characterization of EPO receptor (EPOR) and SCF receptor (KIT)-mediated signal transduction in proerythroblasts revealed a requirement for EPOR, but not KIT, signaling for tyrosine phosphorylation of STAT5 (Tyr694), a downstream target for the cytoplasmic tyrosine kinase JAK2. MAP kinases ERK 1/2 were phosphorylated (Thr202/Tyr204) in response to either EPO or SCF alone, with phosphorylation of ERK1/2 induced predominantly by SCF. We found increased phosphorylation of ERK 1/2 when proerythroblasts were treated with both EPO and SCF. Phosphorylation of protein kinase B/Akt (Ser473), a signaling molecule downstream of phosphatidylinositol 3-kinase (PI3K), was observed following SCF treatment. Treatment with kinase inhibitors targeting JAK, PI3K and MAP kinase kinase (MEK1) during EPO and SCF stimulation revealed that JAK inhibitor AG490 attenuated STAT5 tyrosine phosphorylation, MEK inhibitor PD98059 abolished ERK 1/2 phosphorylation and the PI3K inhibitor LY294002 inhibited Akt phosphorylation. To determine the contribution of specific signaling pathways to synergistic proliferation of proerythroblasts in response to cooperative effects of EPO and SCF, we performed proliferation assays with increasing concentrations of each inhibitor (5 and 50 μM) or DMSO vehicle. We found significant, dose-dependent inhibition of proerythroblast proliferation in response to all three JAK, PI3K or MEK inhibitors (figure 1, *P

Description: Translational regulation plays a central role in cell proliferation, survival and cell differentiation through activation of the target of rapamycin (mTOR) signaling pathway. mTOR controls the phosphorylation status of proteins involved in initiating translational control, including ribosomal S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Recently, the mTOR and phosphoinositide 3-kinase (PI3-K) pathways have been linked through the tumor suppressor complex TSC1/2. The PI3-K target PKB inactivates the TSC1/2 complex which acts as a GAP for the Ras homologue Rheb to suppress mTOR signaling. As the regulation of cell number and cell size are important factors during megakaryopoiesis, we investigated the role of mTOR signaling in thrombopoietin (TPO) induced proliferation and differentiation in primary human megakaryocyte progenitors and in megakaryoblastic MO7e cells. Preincubation of isolated CD34+ cells, primary cultured CD61+ cells and MO7e cells with the mTOR inhibitor rapamycin (10 nM) resulted in highly specific inhibition of TPO-mediated 4E-BP1 (S65) phosphorylation, S6K (T389) phosphorylation and its substrate S6 (S235/236), without affecting PKB (S473) phosphorylation. Activation of the mTOR signaling pathway by TPO was dependent on the PI3-K pathway as LY294002 (10 μM) inhibited phosphorylation of 4E-BP1, S6K and S6. Treatment of MO7e cells with rapamycin inhibited TPO-induced proliferation and cell cycling by reducing cells in S-phase and blocking cells in G1. Rapamycin did not induce apoptosis as measured by cells in sub-G0 phase and by Annexin V expression. Suspension cultures of CD34+ cells treated with rapamycin resulted in a 2.3-fold reduction in overall cell proliferation (p=0.01) and a reduction in the percentage of CD61+ megakaryocytic cells (55.2±7.93 vs 35.0±5.42, p=0.08) generated after 7 days. The mean fluorescence intensity of CD61 and CD42 expressing cells was however not decreased. In addition, polyploidisation levels of the CD61 expressing cells cultured in the presence of rapamycin were not decreased, indicating rapamycin inhibited megakaryocyte proliferation, not differentiation. Further analysis revealed that transforming growth factor β1 (TGF-β1), which inhibits proliferation of megakaryocyte progenitors, downregulated TPO-induced S6K/S6 phosphorylation. Concluding, we have shown that the mTOR pathway is activated by TPO and plays a role in regulating proliferation in megakaryocyte progenitors. Part of the effect of the PI3-K/PKB and TGF-β pathways in regulating proliferation may be mediated by the mTOR/S6K/4E-BP1 signaling pathway.

Description: In the hematopoietic system, TGF-β1 is one of the most potent extrinsic regulators, affecting both early progenitors and committed cells. At the top of the hematopoietic hierarchy, TGF-β1 maintains hematopoietic stem cells (HSCs) in quiescence in vitro through transcriptional regulation of genes encoding proteins important in the cell cycle. We have shown that TGF-β receptor I (TβRI) −/− HSCs exhibit increased proliferative capacity in vitro and that TβRII−/− mice develop a multifocal autoimmune disease, mainly mediated by T-cells (Larsson et al, 2003, Levéen et al 2002). The mechanisms of TGF-β signaling in hematopoietic cells are poorly understood and many target genes of TGF-β signaling remain elusive. In this study we have used global gene expression analysis to investigate whether all TGF-β signaling is mediated by TβRI and II. Furthermore, we asked what target genes are affected upon TGF-β stimulation in normal and TGF-β signaling deficient murine embryonic fibroblasts (MEFs). MEFs were grown with and without TGF-β1 stimulation and proliferation, transcriptional responses and expression analysis were performed. We demonstrate through Western Blot analysis, luciferase reporter assays and cell expansion experiments how these cells lack functional TβRI. Additionally, transcriptional assays show that no other Smad activity is triggered by TGF-β1 stimulation. Furthermore, we demonstrate through quantitative RT-PCR that the inhibitor of differentiation family of genes, known targets of TGF-β signaling, are not affected by TGF-β1 in TβRI−/− MEFs, while wt cells downregulate these genes 4–8.5 fold in response to stimulation. In order to completely exclude alternative receptors outside the TGF-β superfamily and signaling pathways activated through TβRII alone, we performed global gene expression profiling on TGF-β1 stimulated TβRI−/− MEFs with unstimulated TβRI deficient cells as reference. Very few (0.05 %) of the more than 37,000 spots on the microarray had a 〉2 fold differential expression in the two experiments conducted. Similar experiments performed on wt cells resulted in differential expression of between 2.6–3.9 % of the genes printed. From this data we conclude that no signaling affecting gene expression occur in the absence of TβRI in these cells. Additionally we present transcriptional profiles of MEF cell lines that either are normal or are TβRI deficient. By means of cDNA microarray technology, we have identified genes that were differentially expressed when TβRI deficient fibroblasts were compared to wt cells stimulated with TGF-β1. Our results create a data base of 461 significantly differentially expressed (p

Description: Erythropoietin (EPO), the primary cytokine regulator of red blood cell production, acts through binding to its cognate receptor (EPO-R), which is primarily expressed on erythroid precursors. Knockout studies have illustrated a critical role for EPO, EPO-R and the downstream tyrosine kinase JAK2 in embryogenesis as mice lacking any of these components die from a fatal anemia at E13.5. These data suggest that EPO-R and/or JAK2 are required to promote erythropoiesis in vivo. EPO provides mitogenic, differentiative and cell survival signals to erythroid progenitors. We have performed microarray studies to identify target genes regulated by EPO in cell lines and primary cells. We utilized an erythroid cell line (HCD-57), a myeloid cell line stably expressing the EPO-R (Ba/F3-EPO-R), fetal liver cells isolated from E13.5 mice as well as splenocytes isolated from Phenylhydrazine (PHZ)-primed adult mice. Fetal liver cells permit the study of normal erythropoiesis in a fetal setting whereas the PHZ-primed erythroblasts permit analysis of stress erythropoiesis in adult mice. We harvested cells at 1, 8, 12 and 24 hr after EPO stimulation which correspond to immediate early gene induction (1 hr), S phase entry (8 hr) and G2/M (24 hr) time points. RNA was prepared and hybridized to the Affymetrix U74A mouse chip. Data was analyzed and only those genes with statistical significance (p 〈 0.05) were considered for further characterization. Analysis of the 1 hr time points has revealed that six genes are co-regulated by EPO in all four cellular environments. Included within this co-hort are the Suppressor of Cytokine Signaling genes (Cis, SOCS-1 and SOCS-3) and Myc, as well as two novel genes. We compared our datasets with other published analyses. The Williams laboratory has identified an Interferon-Stimulated Gene “ISG” data set corresponding to genes induced by Type I or Type II Interferon’s. We queried our PHZ-primed erythroblast data set against the Williams ISG database. Of the 305 human genes in the ISG database, 218 are expressed on the Affymetrix chip. We searched our dataset for genes that are induced 1.5-fold or greater at 2 of 4, 3 of 4 or 4 of 4 time points. Thirty-four genes are also stimulated by EPO in PHZ-primed erythroblasts including classical IFN-regulated genes such as Interferon-regulator factor-1 (IRF-1), Interferon-stimulated gene-15 (ISG-15), Interferon-induced transmembrane protein 3-like (IFITM-3l), Protein Kinase R (PKR) and Signal Transducer and Activator of Transcription-1 (STAT1). We have previously demonstrated that STAT1 is a negative regulator of murine erythropoiesis utilizing STAT1-deficient mice. We also analyzed immediate early gene regulation in fetal liver cells and PHZ-primed erythroblasts isolated from STAT1-deficient mice stimulated with EPO for 1 hr. These data were compared with the relevant wild type data sets. EPO stimulates the induction of the ubiquitin-like protein, ISG-15 in both wild type and STAT1−/− erythroblasts. Several signaling proteins have been shown to be covalently modified by ISG-15 including STAT1. ISG-15 is removed from ISGylated products by the deubiquitinating enzyme, Ubp43. EPO stimulates a rapid accumulation of Ubp43 in wild type cells, however, EPO fails to induce Ubp43 mRNA in STAT1-deficient fetal liver and PHZ-primed erythroblasts. Experiments are underway to confirm that the mechanism by which STAT1 exerts negative regulation of erythropoiesis is via upregulation of the deubiquitinating enzyme, Ubp43.

Description: The development of hematopoietic stem cells (HSCs) is orchestrated by numerous hematopoietic cytokines, growth factors and chemokines. For the most part these proteins are secreted from bone marrow microenviromental cells, including fibroblasts, endothelial cells and stromal cells, or are displayed on their cell surfaces. HSCs have also been shown to produce numerous cytokines and growth factors. Interestingly, secreted cytokines have also been reported to induce the production of additional cytokines from neighboring cells, suggesting that cytokines drive networks of other cytokines to support hematopoietic cell development. Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis and vasculogenesis, also plays an important role in HSC development, where it acts in an intracellular autocrine fashion to promote cell survival. The secretion of VEGF from endothelial or smooth muscle cells is regulated by extracellular stimuli, inflammatory cytokines and hypoxia. In contrast, it is not clear whether synthesis of VEGF in HSCs is regulated by extracellular signals. Because several early acting cytokines, including TPO, affect VEGF-A expression in hematopoietic cell lines, we hypothesized that TPO could be a regulator of a HSC VEGF autocrine loop. We found that TPO induces VEGF transcripts in primitive marrow derived sca-1+/c-kit+/Gr-1− hematopoietic cells, and that VEGF transcripts are reduced in these cells when derived from Tpo−/− mice. Additional studies determined that TPO induces VEGF expression by increasing the nuclear levels of its primary transcription factor, both in the TPO-dependent primitive hematopoietic cell line UT-7/TPO and in purified sca-1+/c-kit+/Gr-1−marrow cells. Elevation of HIF-1α by TPO is achieved by two different mechanisms; augmented protein synthesis and enhanced stabilization. The latter mechanism is dependent on heat shock protein 90 (Hsp90), as inhibition of Hsp90 function by the specific inhibitor geldanamycin inhibited the TPO-dependent stabilization of HIF-1α. In additional studies we also established that VEGF expression was important for the favorable TPO effect on primitive hematopoietic cells, as blockade of the VEGF receptor with a specific inhibitor, SU5416 (0.1mM), substantially blunted TPO induced growth of sorted single sca-1+/c-kit+/Gr-1−marrow cells in serum-free cultures. Importantly, this inhibitor does not affect TPO induced phosphorylation of Jak2, ERK and AKT in UT-7/TPO cells even at a 100-fold higher concentration. Along with our previous findings that TPO affects Hox transcription factors that regulate HSC proliferation, these data contribute to our growing understanding of the mechanisms by which a hormone can influence HSC development.

Description: Erythropoietin (Epo) is a key molecule in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated Epo-receptor. We have recently identified Cbp (Csk binding protein, a negative regulator of Src) as a novel interactor with Lyn. The proline-rich region of Cbp associates with the SH3 domain of Lyn, which then phosphorylates Cbp on multiple tyrosine residues forming additional binding sites for the Lyn SH2 domain. Phosphorylated Y314 of Cbp, in turn, recruits the negative regulators Csk/Ctk, which phosphorylate Lyn and down-regulate its kinase activity. A phosphotyrosine-specific yeast two-hybrid system we developed, confirmed that the SH2 domain of Csk associated with Y314 of Cbp. Significantly, another well characterized negative regulator, SOCS1, also bound this phosphorylated tyrosine residue, resulting in elevated ubiquitination and degradation of Lyn. Thus, Cbp co-ordinates a two step down-regulation of Lyn. In Epo-responsive J2E cells, Cbp is rapidly phosphorylated by Lyn and the enzymatic activity of Lyn is suppressed by Csk/Ctk after 10 minutes of Epo stimulation. Subsequently, SOCS1 is involved in the turn over of Lyn protein two hours post-Epo induction. These data demonstrate for the first time a two phase process regulated by Cbp for inactivating and degrading Lyn after Epo stimulation.

Description: Background: The colony-stimulating factors (CSFs) are widely utilized to prevent neutropenic complications in both adults and children, but randomized controlled trials in the pediatric setting have varied considerably in size, methodology and patient population. A systematic review and meta-analysis was conducted to more definitively assess the impact of prophylactic CSFs on the risk of febrile neutropenia (FN) in pediatric oncology patients. The rates of documented infection (DI) and duration of severe neutropenia (ANC

Description: Between 05/98 and 06/04, 15 consecutive patients with FA received hematopoietic stem cell transplants (SCT) from alternative donors at our Center. There were 7 males and 8 females aged 5 to 24 years (median 11.5). Hematologic diagnoses included aplastic anemia (AA) (N=5), myelodysplastic syndrome (MDS) in RAEB (N=4), RAEBT (N=1) or acute myelogenous leukemia (AML) (N=5). High risk features included: Age 〉 20 years (n=4), prior multiple transfusions (n=11), prior androgen treatment (n=12), prior infections (n=10), or advanced MDS or AML (n=9). Eight pts had related mismatched donors transplants with respective matching at 3/6 (6/10), 4/6 (6/10), 4/6 (7/10) (n=2)), 5/6 (8/10) (n=3) and 5/6 (9/10) HLA-antigens. Seven pts had unrelated donors transplants with respective matching at 5/6 (7/10), 5/6 (8/10) (n=2), 5/6 (9/10) and 6/6 (10/10) (n=3) HLA-antigens. Cytoreduction included single dose total body irradiation (SDTBI) (450 cGy), fludarabine (Flu) (30 mg/m2 x 5) and cyclophosphamide (Cy) (10 mg/Kg x 4). Immunosuppression included rabbit anti-thymocyte globulin (Thymoglobulin) and tacrolimus for all patients. Grafts were G-CSF mobilized CD34+ and E-rosette negative (E-) peripheral blood stem cell transplants for 12 pts and soybean agglutinin negative (SBA-) and E-rosette negative marrow transplants for 3 pts. Cell doses of the grafts were 1.5 – 29.6 x 106 CD34 cells/Kg and 0 – 26 x 103 CD3 cells/Kg. As evidenced by RFLP or FISH, all 15 evaluable pts were fully engrafted and complete chimeras. Fourteen pts were evaluable for graft-versus-host disease (GvHD). GvHD of the skin and of the gut was suspected in two pts but resolved completely prior to immunosuppressive treatment. With a median follow-up of 2.5 years (range 0.2–6), 13 of 15 pts are alive and 11 of 15 are alive disease-free. There were two deaths: one pt died from sepsis/ARDS at 2 months post SCT and one pt from pneumonitis/ARDS and EBV-infection 6 months post SCT. Three pts relapsed (MDS-RAEB x 1 – AML x 2): One pt relapsed 7 months post transplant, received a 2nd transplant from the same donor following busulfan and Flu and is alive, disease-free 18 months post SCT, while the other two pts are awaiting a second SCT. In summary, this cytoreductive regimen used with T-cell depleted stem cell transplants from unrelated or HLA-mismatched related donors for the treatment of high risk patients with Fanconi anemia, results in rapid hematopoietic engraftment and lymphohematopoietic reconstitution with minimal GVHD and a high disease-free survival.

Description: We recently reported that Tyk2 was essential for IFN-a-induced B lymphocyte growth inhibition, although Stat1 is not required for this IFN-a-mediated inhibition. This means that other signaling molecules besides Stat1, and which are activated by Tyk2, are thought to transduce the IFN-a signal inhibiting B lymphocyte growth. We performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Rack-1, originally described as a receptor for activated C kinase beta, associated with Tyk2. Receptor for activated C kinase (Rack)-1 is a protein kinase C interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase C gamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alpha/beta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudo-kinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N-terminus and one in the middle portion (a.a.138–203) of Rack-1. In addition, not only Tyk2 but other Jak kinases associate with Rack-1, and each Jak activation causes the phosphorylation of Tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated from cytoplasm or membrane toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.

Description: Background: In October 2003, six months after approval of the first drug-eluting coronary stent (CYPHER™ stent), the FDA reported 50 stent-associated hypersensitivity reactions. In November 2003, FDA officials reported that these reactions were probably due to concomitantly prescribed medications. Methods: All adverse device event reports for CYPHER stent associated hypersensitivity- reactions in the FDA’s adverse event reporting system, the published literature, or from RADAR, between April 2003 and June 2004 were reviewed. Causality was evaluated using World Health Organization criteria. Results: Review of FDA reports (n=2,607) identified 161 persons with hypersensitivity symptoms. Fewer than 20 hypersensitivity cases were reported monthly, except for 43 and 42 reports in October and November 2003. Symptoms began within 2 days of stent implantation (21%), between 2 and 7 days (43%) and between 7 and 14 days (19%) following stent implantation and persisted for less than 8 days for 17%, 8–30 days for 29% and 〉 30 days for 55%. Findings included rash (80%), itching (28%), hives (19%), dyspnea (12%), fever (12%), chest pain (8%), anaphylaxis (7%), joint pain or swelling (7%) and high or low blood pressure (5%). Among persons with rashes, 25% covered the entire body, 21% developed hives, 7% had localized maculopapular eruptions and 4% developed blisters or desquamation. Treatment included emergency interventions (26%) or hospitalization (19%). Outcomes included permanent disability (5%) or death (2%). Aspirin or clopidogrel was discontinued at the time of hypersensitivity onset for 42. Using WHO criteria, clopidogrel, aspirin, ticlopidine and/or the stent itself were classified as possible causes for 〉 85% of cases. Of 150 events possibly caused by the CYPHER stent, 35 persisted for 〉 30 days. For 7 probable CYPHER-stent induced hypersensitivity cases identified by RADAR (n=4), the FDA (n=2) or the literature (n=1), symptom onset was 〈 5 days (n=3) and at 3 weeks, 1 month, 4 months, and 7 months after stent implantation. Findings included rash (n=4), hives (n=1), blisters on both hands (n=1), dyspnea (n=2), eosinophilia (n=1), elevated IgE levels (n=2) and Gallium-67 scan findings at the stent (n=1). Findings did not abate with thienopyridine discontinuation (n=5). Outcomes included resolution of symptoms with hospitalization (n=1) or corticosteroids (n=2), persistent angina (n=1) and subsequent occurrence of fatal cardiac events, with autopsy identification of eosinophilic infiltrates at the stent site (n= 2 patients). Conclusion: Rash, hives, dyspnea and catastrophic cardiac events may represent local and systemic hypersensitivity reactions from the CYPHER-stent. Reporting of these events decreased by 〉 90% following a FDA advisory that suggested that skin reactions were unrelated to the stent. However, our findings highlight the importance of continued vigilance for hypersensitivity reactions that may represent an early manifestation of a catastrophic hypersensitivity event.

Description: Background: Cancer is associated with thrombosis, but the frequency of thromboembolism in hospitalized cancer patients receiving contemporary chemotherapy regimens is not known. We investigated the frequency of arterial and venous thromboembolism in hospitalized cancer patients receiving active therapy (as identified by the presence of neutropenia) and characterized its association with in-hospital mortality. Methods: We conducted a retrospective cohort study using the discharge database of the University HealthSystem Consortium. This included 66,106 adult neutropenic cancer patients with 88,074 hospitalizations between 1995 and 2002 at 115 academic medical centers. Patients were identified using ICD-9-CM codes that contained at least one diagnosis of malignant disease and agranulocytosis. Patients with thromboembolism were identified using codes for venous thrombosis, pulmonary embolism, arterial embolism, acute cerebrovascular disease, and acute coronary arterial disease. The association of VTE with clinical variables was studied in univariate analysis and in a multivariate logistic regression model. The chi-square test was used to compare categorical variables, and Cochran-Armitage test to determine trend. Results: Thromboembolism was reported in 5,272 patients (8%), with 5.4% patients developing venous and 1.5% arterial thromboembolism during the first hospitalization. There was a significant association between the occurrence of venous and arterial thromboembolism (OR 1.73, 95%CI, 1.38–2.16). Venous thromboembolism was more frequent in patients with metastatic disease (OR, 1.23, 95% CI 1.13–1.34), but arterial thromboembolism was not (OR, 0.59, 95% CI, 0.51–0.69). In-hospital mortality was significantly greater in patients with venous (OR 2.01, 95% CI 1.83– 2.22) or arterial thromboembolism (OR 5.04, 95% CI, 4.38–5.79), even in patients without metastatic disease. Patients with lymphoma or leukemia accounted for one-third of venous events and one-half of arterial events. Clinical variables most frequently associated with thromboembolism in a multivariate logistic regression analysis were age ≥ 65 years, primary sites of cancer including lung, gastrointestinal, gynecologic and brain, length of stay ≥ 10 days, and comorbidities including infection, pulmonary and renal disease, and obesity. From 1995 to 2002, there was a 36% increase in venous and a 124 % increase in arterial events (P for trend

Description: To study transplanted unperturbed and mobilized long-term hematopoiesis after selection with an alkylating agent, bone marrow (BM) from 5 C57BL/6J mice was pooled, repeatedly transduced with retroviruses encoding the alkylating agent resistance protein O6-Methylguanine-DNA and enhanced green fluorescent protein (eGFP) as an easily traceable marker. Between 1 to 9x105 transfected BM cells were transplanted into 15 myeloablatively irradiated sex-mismatched C57BL/6J mice. Subsequently, 3 to 4 selection rounds with BCNU/O6-BG were carried out, enriching eGFP marked hematopoiesis in these mice up to 70–90%. Between 1 and 7x107BM cells of different mice were transplanted according to marrow location into groups of 5 sex-matched Bri44[1] mice. Two mice each received BM from the hind limbs, two from the pelvis and one received cells from the spleen, only, respectively. Altogether the study comprised 15 groups divided into 6 female and 9 male groups. Of these, 4 male and 3 female groups received 3 HSC-mobilization courses with G-CSF at intervals of 2 months starting 3 month after transplantation. Hematopoiesis in the other fraction remained unperturbed. During the observation period of 11–14 months in these tertiary recipients, repeated FACS analyses as well as linear amplification mediated (LAM) PCRs were carried out to track the clonal contributions. A decrease in the percentage of eGFP expressing marked hematopoiesis was observed in most cases. However, eGFP expression never disappeared altogether and could still be detected in the different hematopoietic lineages and successfully sorted for further analyses by MoFlo (Dako-Cytomation). Assessment of the clonal status of the Bri44 by LAM-PCR displayed interesting results. In some mice a decline in clone numbers was observed, whereas clone numbers remained stable in others. Tertiary transplantation with long-term follow-up indicates that this observation may be related to the transplantation of limited long-term repopulating clone numbers and progenitor cell exhaustion over time.

Description: Recent studies in the NOD/SCID model have shown improved engraftment of SCID-repopulating cells and higher levels of engraftment in the secondary transplantation when cells were administered by intramarrow (IM) versus intravenous (IV) injection suggesting that direct injection into the marrow cavity may be beneficial for stem cell engraftment in a clinical setting. To study whether IM injection was feasible and would result in improved engraftment in a clinically relevant large animal model, we compared IM vs IV injection in our competitive repopulation assay in baboons. Enriched CD34+ cells were split into 2 equal fractions and transduced with either a GFP- or YFP-expressing vector. Pretransplant transduction efficiencies and expansion of CD 34+ cells were similar in both fractions. One fraction was then infused into the marrow cavity of the right femur and the other fraction was given intravenously. Three baboons received gene-modified CD34+ enriched autologous bone marrow cells after myeloablative radiation. Peripheral blood granulocyte marking levels showed peaks at 2–3 weeks after transplantation and decreased thereafter. In all three monkeys, marking levels of IM injected cells (GFP) were lower than marking levels of IV injected cells (YFP) early after transplantation up to 7 weeks. However, in two of the three monkeys, GFP marking increased steadily after 2 months resulting in higher marking levels from IM injected cells. The trend sustained up to the last follow-up of nine months after transplantation, marking levels being 25.5% and 7.4% from IM and IV injected cells, respectively, in M00228. This pattern was recapitulated in the marking of bone marrow cells of the two animals. GFP (IM) and YFP (IV) marking levels of bone marrow cells from non-injected bone were 24.2% and 33.9%, respectively, at 1 month, 7.9% and 4.6% at 3 months, 19.1% and 12.6% at 6 months after transplantation in M00228. In addition, the GFP marking of the bone marrow cells from the injected bone was higher than that of the BM cells from non-injected bone while YFP marking level was similar. In conclusion, our data suggest that direct intramarrow injection of CD34+ cells may lead to improved engraftment of long-term repopulating cells. Clonal analysis is currently under way to determine the clonal pattern of the differentially marked repopulating cells.

Description: Forty patients with relapsed or refractory Hodgkin’s disease (HD) underwent allogeneic stem cell transplantation (allo-SCT) following a fludarabine-based conditioning regimen from an HLA-identical sibling (n=20) or a matched unrelated donor (n=20). The median age was 31 years (range 18-58). The median number of chemotherapy regimens received prior to allo-SCT was five (range 2-9). Thirty (75%) and thirty (75%) patients had received prior radiotherapy or a prior autologous SCT, respectively. The median time to progression after autologous SCT was nine months (3–52). Disease status at SCT was refractory relapse (n=14) or sensitive relapse (n=26). The conditioning regimens employed were fludarabine (25 mg/m sq IV x 5 days)-cyclophosphamide (1 g/m sq IV x 3 days) ± antithymocyte globulin (30 mg/kg IV x 3 days) (FC±ATG) (n=14), a less intensive regimen, and fludarabine (25 mg/m sq IV x 5 days) -melphalan (70 mg/m sq IV x 2 days) (FM) (n=26), a more intensive one. The two groups had similar demographics and prognostic factors. Chimerism studies indicated 100% donor-derived engraftment in 26/26 (100%) FM patients and in 9/13 (69%) evaluable FC±ATG patients. Day 100 and cumulative (18-month) transplant-related mortality (TRM) were 5 % and 22%, respectively for the whole group. There was a nonsignificant trend towards a lower cumulative TRM in the FM group (18% vs. 30% at 18 months, p=0.2). The cumulative incidence of acute (grade II-IV) GVHD was 38%. The cumulative incidence of chronic GVHD at 18 months was 69%. There was a trend for a lower relapse rate after the occurrence of GVHD, however, this was not statistically significant (hazard ratio 0.8; p= 0.6). Progression rates were similar in the FM and FC patients (53% vs. 57% respectively at 18 months, p=0.4). However, disease progression occurred later in FM patients (range 2–34 months) than in FC patients (range 0.7–13 months). In addition, with comparable follow-up time after progression, the FM group experienced a lower death rate after progression. Twenty-four patients (60%) are alive (fourteen in complete remission) with a median follow-up of 13 months (4–78). Sixteen patients expired (TRM n=8, disease progression n=8). FM patients had significantly better overall survival (73% vs. 39% at 18 months; p=0.03), and a trend towards better progression-free survival (37% vs. 21% at 18 months; p=0.2). We conclude that allo-SCT with fludarabine-based, less intensive conditioning from matched related and unrelated donors are feasible in high-risk HD patients with a low TRM. The intensity of the preparative regimen affects survival.

Description: HIV-based lentiviral vectors (LV) have become powerful tools for in vivo gene transfer and gene expression in non-dividing cells after local injection. However, the potential of in vivo bone marrow stem cell gene transfer by “in situ” bone cavity injection has not been well unexplored. This in vivo approach could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo hematopoietic stem cell (HSC) gene transfer. To determine if intra bone marrow (iBM) injection of LV can efficiently transduce BM stem cells, a 3rd-generation self-inactivating LV, containing eGFP regulated transcriptionally by a CMV promoter, was used to inject intra-femorally into adult Boy/J Ly45.1 mice (at 2 x106 IU/mouse). Blood counts were normal in all LV-injected mice (n=4) and buffer-injected mice (n=4). GFP-expressing peripheral blood leukocytes (PBL) were observed in both myeloid (up to 4%) and lymphoid subpopulations (up to 2%) 3-month post injection. To evaluate gene transfer and transgene expression in multi-potential progenitors, the colony-forming cell (CFC) assay was performed using low-density bone marrow 4-months post injection, resulting in 4–5% GFP+ CFU-granulocyte macrophage (CFU-GM) and CFU-granulocyte, erythroid, macrophage and megakaryocyte (CFU-GEMM). This was consistent with the 4-color FACS analysis data demonstrating that up to 3.7% of HSC/progenitors (Lin−c-kit+Sca1+ cells) from LV-injected mice expressed GFP transgene 4-months post injection. To further evaluate HSC gene transfer, BM from injected mice was transplanted into lethally irradiated adult C57/Bl6 (Ly45.2) mice. Four months later, successful engraftment was demonstrated in all BM recipients with 97–99% donor-derived cells in PBL and 91–93.3% in BM. Moreover, significant levels of GFP+ CFU were observed in all recipients ranging from 8.4 ± 2.3% to 17.7 ± 4.2%. To further study the LV-mediated gene transfer profile in HSC, mesenchymal progenitors and systemic distribution by intra-femoral injection, molecular analyses are in progress including real-time QPCR and clonality analysis by LAM-PCR. Our results indicate successful LV-mediated gene transfer into HSC by iBM injection. These data would potentially open a door to a novel approach for treatment of human diseases, but also provide a new tool to study adult stem cell plasticity and the nature of hematopoiesis.

Description: Methotrexate (MTX) is a universal component of chidlhood ALL therapies and its conversion to long chain polyglutamates (PG) by folylpoly-γ-glutamate synthetase (FPGS) is essential for its antileukemic acitivity. Expression of FPGS appears to be controlled by tissue/lineage specific and proliferation-dependent mechanisms. Levels of FPGS mRNA, protein, and enzyme activity are 2-3 fold higher in B-precursor (Bp) ALL cells when compared to T-lineage ALL, and these differences correlate with intracellular accumulation of long chain MTX-PG and lymphoblast sensitivity to MTX. To characterize these lineage differences in FPGS expression between B and T lymphoblasts we examined its mRNA expression in Nalm6 (Bp-ALL) and CCRF-CEM (T-ALL) cells during cellular growth and cell cycle checkpoints. During early exponential growth (24 hrs), FPGS expression was 6-fold higher in Nalm6 when compared to CCRF-CEM but decreased significantly after 72 hrs while it was unchanged in CCRF-CEM cells. During G1/G0 phase we found that FPGS expression was 15-fold higher in Nalm6 when compared to CCRF-CEM cells. Taken together, these data suggest that during proliferation and cell cycle progression FPGS gene expression is regulated differently in Bp-ALL and T-ALL cells. To determine whether this lineage-specific regulation occurs at the transcriptional level we performed nuclear run-on assays. We found that FPGS mRNA transcription initiation rate was 1.7-fold higher in Nalm6 when compared to CCRF-CEM cells, indicating that differences in promoter regulation lead to the observed lineage differences in FPGS expression in Bp- vs. T-ALL. We then used a methylation specific PCR assay to investigate the methylation status of the FPGS proximal promoter region from both Nalm6 and CCRF-CEM cells. Our data indicate that the FPGS proximal promoter region is unmethylated in both cell lines and therefore can not explain the observed lineage-specific differences. 5′-RACE experiments demonstrated that Nalm6 and CCRF-CEM have the same FPGS transcriptional start sites, but a reporter gene assay indicated that the minimal promoter region that directs FPGS transcription in CCRF-CEM cells is insufficient to drive FPGS mRNA transcription in NALM6 cells. In order to identify potential regulatory regions directing FPGS transcription in Bp-lymphoblasts, we used DNaseI hypersensitivity assays. We identified a hypersensitive region located 8.5 kbp upstream to exon 1 in Nalm6 cells suggesting that tissue-specific regulatory elements responsible for lineage-specific FPGS expression in Bp-ALL cells may be localized within this region. Finally, we detected reduced levels of FPGS mRNA expression in RCH-ACV (Bp-ALL, t(1:19)/E2A-PBX1) and REH (Bp-ALL, t(12:21)/TEL-AML1) cells expressing chromosomal translocated fusions when compared to control (Nalm6). To characterize the molecular basis of the E2A-PBX1 and TEL-AML1 interactions with FPGS mRNA expression in Bp lymphoblasts we used antisense and RNAi technology to downregulate these two genetic fusions. Our data lead us to hypothesize that in addition to lineage-specific regulatory differences in FPGS expression, molecular mechanisms associated with non-random translocations may alter FPGS mRNA expression and influence MTX sensitivity in Bp-ALL lymphoblasts.

Description: DVd in combination with Thalidomide (T) and the appropriate supportive care measures resulted in a high response rate (88%) as well as an improved quality response (50% CR & NCR) similar to what is achieved with high dose therapy. Immunomodulatory drugs (IMiDs) are potent T derivatives. R is 50 to 2000 times more potent than T in stimulating T-cell proliferation triggered via the T-cell receptor, and 50 to 100 times more potent than T in augmenting IL-2 and IFN-a production. A recent phase I trial showed responses of at least 25% reduction in paraprotein in 17 (71%) of 24. We therefore initiated a phase I/II trial to define MTD of R in combination with DVd, then we proceeded to expand the MTD dose level to evaluate the efficacy and safety of the combination in patients with Rmm. SWOG criteria were used to assess response, and NCR was defined as a decrease of the M-Protein by 〉90%. Refractory patients were defined as those patients progressing on active therapy. R was started a week prior to DVd in cycle 1 to evaluate different coagulation parameters, from there on R was started on day 1 of therapy. The regimen was given as follows: on day 1 D was given at 40 mg/m2 IVPB; V at 2 mg IVP; d at 40 mg PO daily X 4 days; R was started at 5 mg a day for 21 days with one week off. A standard phase 1 dose escalation of R was performed to identify the MTD. 3 pts were enrolled at each dose level, with up to 6 pts assigned to each dose level, depending on DLT. DVd was repeated q 4W, for a minimum of 4 cycles & 2 cycles after best response. Pts were maintained on R +/− prednisone 50 mg QOD. All patients received amoxicillin, acyclovir and aspirin 81mg prophylactically. 25 pts Rmm pts are enrolled with 21 evaluable for toxicity and mature data available for response on 21 pts (refractory: 15 (71%); relapsed: 6 (29%). 17/21 patients were stage 3, median age of 62 ± 9 years, baseline b2 microglobulin level (mean 5.04 ± 2) and serum albumin (mean 3.4 ± 0.7). 14/21 patients failed T containing regimens. The DLT was sepsis/septic shock that occurred at dose level 3 (R 15mg) with two of the patients developing non neutropenic sepsis. The MTD for R was defined at 10mg. Three patients started therapy with a neutrophil count 〈 500/mL and or platelet counts 〈 50k/mL; all 3 patients were responders. There was one grade 4 hyper-coagulation event in the form of a PE that has recovered. This event occurred in a refractory patient with renal failure performance status of 3 who achieved CR after 2 cycles. In the expanded cohort there was 2 grade 3 neutropenia & one grade 3 neuropathy requiring dose reductions of R and D in each pt with resolution of the neutropenia and neuropathy. 3/21 (14%) patients achieved CR, 4/21 (19%) NCR. All CR+NCR patients (33%) are refractory patients. An additional 7 pts achieved PR, and 6 SD 5 of whom showing greater than 25% decrease in the m-protein. All patients except for 4 achieved 〉 25% reduction of the m-protein after one cycle of therapy and 3/4 after 2 cycles. R at 10mg is the MTD in combination with the DVd in RMM. DVd-R is an extremely effective regimen with a SWOG response rate 〉66%, CR+NCR of 33% in refractory stage 3 patients with minimal toxicity.

Description: Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate (THF) required for the synthesis of thymidylate and purines. Methotrexate (MTX) acts as a tight-binding inhibitor of DHFR and remains an important chemotherapeutic agent for treatment of leukemias and lymphomas. Increased DHFR confers resistance to antifolates in target cells. A previously reported single nucleotide polymorphism (SNP) 829C/C→829T/T (829C→T) found in the 3′- untranslated region of DHFR gene transcript (between the first and second polyadenylation site) was associated with higher expression of the DHFR transcript. The SNP was identified in 5.4% of the cases and 6.0% in the controls of Japanese patients with childhood leukemia/lymphomas (Goto et al. 2001, Clinical Cancer Research, Vol. 7, 1952-1956). The objective of the present study was to determine the role of the 3′ UTR SNP 829C→T in DHFR gene expression, DHFR protein level and resistance to MTX. The mutation 829C→T in the 3′ UTR of wild type DHFR was introduced by site directed mutagenesis and the mutant cDNA expressed in DHFR deficient CHO cells (DG-44), wild type DHFR and vector alone constructs were also transfected into DG44 as controls. After two weeks of selection in G418 containing media, several well-isolated surviving colonies were picked and expanded as cell lines in media containing G418. Real-time quantitative PCR was used to compare mRNA and genomic DNA level of the clones while Western blotting was used to compare the protein levels. MTX cytotoxicity assay was carried out in media lacking thymidine. Clones expressing the mutant 829C→T showed greater than two fold enhanced expression of DHFR transcripts as compared to wild type clones. Corresponding to the high mRNA levels, an increase in DHFR protein level was observed in the mutant clones without an increase in DHFR gene copy number. Cytotoxicity studies showed that cell lines with increased levels of DHFR were significantly more resistant to MTX than cells with wild type 3′ UTR. Of interest clonogenic efficiency of the mutants in medium lacking thymidine was greater than wild type and was directly proportional to the level of DHFR expressed in the clones. This study demonstrates that when SNP 829C→T is introduced in the 3′ UTR of wild type DHFR, the expression of the DHFR mRNA is enhanced with a corresponding increase in the protein level. The presence of a SNP 829C→T in patients with ALL may contribute to treatment failure, as MTX is a key drug in curative regimen for this disease. Future studies are directed toward determining the abundance of this SNP in other populations, and the correlation between this SNP and clinical methotrexate resistance and or decreased MTX toxicity.

Description: In newly diagnosed multiple myeloma (MM) patients, the combination melphalan, prednisone and thalidomide induces a fast tumor response with a high complete remission rate. In a prospective randomized trial, we compare the efficacy and toxicity of oral MPT and MP. An interim analysis was conducted after the first 200 newly diagnosed myeloma patients, median age 72, range 56–85, entered the study, between January 2002 and June 2004. At present, 116 patients were evaluated for toxicity and 102 patients for response on an intent to treat basis. The MPT regimen included 6 monthly courses of oral melphalan 4 mg/sqm and prednisone 40 mg/sqm for 7 days every month plus thalidomide 100 mg/day continuously until any sign of progressive disease or relapse. The dose of thalidomide was reduced to 50% when grade II toxicity occurred, and suspended for any grade III. On December 2003, the protocol was amended and enoxaparin prophilaxys was added. The MP regimen was as MPT without thalidomide. The end points of the study were: response, EFS, OS and toxicity. The response rate among patients who received MPT was: 25.9% immunofixation negative CR (CR), 5.5% immunofixation positive near CR (nCR) 48.2% partial remission (PR) (M-protein reduction 50–99%), 9.3% stable disease (SD) (M-protein reduction 0–49%) and 11.1% progressive disease (PD). The response rate after MP was 4.2% CR, 0% nCR, 43.6% PR, 23% SD and 29.2% PD. Response was followed by significant improvement of performance status, skeletal pain, anemia and transfusion requirement. After a median follow up of 15 months, 38 patients relapsed: 11 (29%) after MPT and 27 (71%) after MP. The EFS @ 26 months was 67.8% for MPT and 32.4% for MP (P

Description: In a recent two year period only 14% of platelet aggregation tests performed on 230 patients suspected of having heparin induced thrombocytopenia (HIT) were positive. The test (control platelet-rich plasma 0.14 mL, fresh heated patient serum 0.22 mL plus heparin 0.04 mL (1 Unit/mL final concentration) was performed using suitable controls on a commercial 4 channel aggregometer. 20% or greater increase in light transmission was a positive result. We then started a prospective study in which three different tests were done on the serum of each of 96 consecutive Hines Veterans Affairs Hospital patients who were clinically suspected of having HIT. The three tests were: platelet aggregation (PA) (same method as in the earlier study), C14 serotonin release (standard washed platelet method-SRA) and ELISA (GTI-PF4, Waukeshaw, Wisconsin), all performed on the same serum sample. The SRA and ELISA tests were performed on serum stored at −70°C. Blinded to the test results, a clinical score was determined from the record as HIGH (30% or greater fall in platelet count with recovery within 15 days off heparin), MEDIUM (same criteria but other cause present, or failure to recover platelet counts on continued heparin therapy) or LOW probability (failure to fall at least 30% or to recover from 30% or greater fall within 15 days observation). Results of the clinical scoring and laboratory tests were as follows: Clinical Probabilities: High (n=23), Medium (N=33), Low (n=40). Of the 56 who were High or Medium probability, 41% were High and 59% were Medium probability. Analysis of the results of the laboratory tests for sensitivity and specificity for each category revealed the following: Clinical Probability PA SRA ELISA Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity High and Medium (n=56) 19.6% 97.5% 10.7% 97.5% 10.7% 90% High (n=23) 30.4% 93.1% 17.4% 95.9% 8.7% 89% Conclusions: Sensitivity of the PA test was greater than either of the other two tests, with specificities for the PA and SRA being nearly equal. The ELISA test was relatively insensitive and less specific than then other two.

Description: Idiopathic autoimmune thrombocytopenia (ITP) is an autoimmune disease characterized by autoantibody-mediated platelet destruction. The major platelet antigens that autoantibodies are directed against in ITP are GPIIbIIIa and GPIbα. Intravenous immunoglobulin G (IVIG) is a treatment used in ITP; however, some patients are refractory to this treatment and the mechanisms of action of IVIG in this disease are not fully understood. We tested the hypothesis that IVIG may not be equally effective in preventing ITP caused by GPIIbIIIa versus GPIbα anti-platelet antibodies. Thrombocytopenia was induced in Balb/c mice using three monoclonal antibodies against either mouse platelet GPIIbIIIa (JON1, 2, −3) or against GPIbα (p0p 3, 4, −5). Mouse platelet counts were performed daily from day one (pre-treatment) until day five using a flow cytometric assay. The efficacy of IVIG in inhibiting monoclonal antibody-induced platelet destruction in mice was assessed by intraperitoneal injection of IVIG (2 g/kg) 24-hours before anti-platelet antibody injections (7 mg/mouse, i.v.) on day two. We found that pre-treatment with IVIG resulted in a significantly higher platelet count on day three in all groups of anti-GPIIbIIIa-injected animals (n=3/antibody; for JON1, P=0.002; JON2, P=0.015; JON3, P=0.002) as compared to albumin-treated controls. In contrast, IVIG failed to prevent platelet clearance and thrombocytopenia on day three in two anti-GPIbα antibody-treated groups (p0p3 and p0p5, n=3/antibody), but did inhibit platelet destruction in mice that were injected with p0p4 antibody (P=0.0003). Our results indicate that the efficacy of IVIG treatment in preventing ITP induced by these anti-platelet antibodies is affected by their antigen specificities. These data also suggest that patients with ITP mediated by GPIbα may be less responsive to IVIG treatment than those with ITP mediated by GPIIbIIIa antibodies.

Description: The role of serotonin (5-hydroxytryptamine, 5-HT) on the regulation of blood stem cell proliferation and thrombopoiesis has not been recognized until 1996, when we reported that serotonin has a mitogenic effect on murine megakaryocytopoiesis via 5-HT2 receptors (Yang et al, Blood Coagul Fibrin 1996). Our study also indicated that the uptake ability of serotonin is well established in human megakaryoblasts (Yang et al, Int J Hematol, 1996). 5-HT 2A, 2B and 2C receptors were identified on human megakaryocytes and serotonin also promoted human megakaryocytopoiesis via these receptors (Yang et al, Blood, 2001; 2002 suppl). Thus, we established a new concept that serotonin is a growth factor for megakaryocytopoiesis (Yang et al, Blood, 2003 suppl). We further investigated the role of serotonin on human hematopoietic stem cells, bone marrow stromal cells and platelet formation. Serotonin (200 nM) significantly enhanced TPO, SCF plus FL -induced the ex vivo expansion of CD34+ cells, CD34+38- cells, CD41+61+ cells, CFU-GEMM and CFU-MK from cord blood CD34+ cells (MACS) (n=25) at day 8 (P

Description: CBFβ-SMMHC, encoded by the inv(16) or t(16;16) translocations in approximately 8% of acute myeloid leukemia (AML) cases, is a fusion protein containing amino acids 1-165 of the 182 residue core binding factor β (CBFβ) and the rod domain of smooth muscle myosin heavy chain (SMMHC). The CBFβ domain of CBFβ-SMMHC retains the ability to interact with AML1/RUNX1. The SMMHC domain both mediates multimerization and interacts directly with corepressors, including mSin3A. CBFβ-SMMHC inhibits the expression of AML1-regulated genes, by sequestering AML1 in multimeric complexes and by directly repressing AML1-regulated genes. CBFβ-SMMHC was previously found to slow G1 to S cell cycle progression in hematopoietic cell lines, reflecting repression of AML1-regulated genes required for cell cycle, including cyclin D3. This effect was overcome be exogenous c-Myc or cdk4. In this study, murine marrow or human CD34+ cells were transduced with retroviral or lentiviral vectors, respectively, expressing CBFβ-SMMHC or two mutant variants. CBFβ-SMMHC reduced murine or human myeloid cell proliferation 3- to 4-fold in liquid culture, during a period when control murine cells accumulated 5-fold and human cells 20-fold. CBFβ-SMMHC decreased the formation of myeloid, but not erythroid, colonies 2- to 4-fold, and myeloid colonies expressing CBFβ-SMMHC were markedly reduced in size. Lack of effect on erythroid colonies reflects their lack of expression of AML1. The mutant variant CBFβ-SMMHC(Δ2-11) does not bind AML1 due to a deletion near its N-terminus, and CBFβ-SMMHC(ΔACD) does not multimerize or efficiently bind corepressors due to a 28 residue deletion near its C-terminus. Neither of these mutants, which were expressed at levels similar to wild-type, slowed proliferation or reduced myeloid colonies. CBFβ-SMMHC increased the G1/S ratio in wild-type murine and human progenitors. Proliferation was still slowed in p15(−/−) murine marrow cells transduced with CBFβ-SMMHC, suggesting that additional mutations, such as activation of growth factor receptors and consequent c-myc induction, are required in primary AMLs to allow enhanced proliferation. AML1-ER(T), which contains full-length AML1 and accelerates G1 to S progression in cell lines when activated by 4HT, had an effect opposite to CBFβ-SMMHC, stimulating proliferation of murine or human myeloid progenitors 2-fold. In summary, CBFβ-SMMHC inhibits the proliferation of myeloid progenitors dependent upon inhibition of AML1 and integrity of its Assembly Competence Domain. Targeting the CBFβ-SMMHC ACD or its CBFβ domain may uncover novel therapeutics useful for AML cases expressing this oncoprotein. Furthermore, these findings support a model we have proposed previously which states that mutations which accelerate G1 are required during leukemogenesis by CBFβ-SMMHC and other CBF oncoproteins. Finally, our results lend support to the conclusion that AML1 participates in the regulation of normal myeloid stem-progenitor cell proliferation. Exogenous AML1 may therefore be useful for expansion of hematopoietic stem-progenitor cells.

Description: The main cause of thrombosis, a major complication of essential thrombocythemia (ET), is the high platelet count. In contrast, thrombosis is rare in reactive thormbocytosis (RT), indicating that thrombocytosis can not be the sole cause of thrombosis. Moreover, thrombosis occurs in ET even after reduction of the platelet count. Tissue factor (TF) is the most potent activator of coagulation. Monocytes are a main source of TF and monocyte TF is increased in various disorders including solid tumors and polycythemia vera that are associated with increased incidence of thrombosis. The decreased incidence of thrombosis that was reported recently in ET after treatment with hydroxyurea compared to anagralide was attributed to a decrease in leukocytes that occurs only after hydroxyurea. Based on this information, we studied the capacity of monocytes from patients with ET to generate TF. Peripheral blood mono-nuclear cells (PBMC) were isolated on Ficoll-hypague from 14 patients with ET and 10 with RT due to iron deficiency. Monocytes were counted by anti CD14 and were the same in both groups (18%). Monocytes were purified from lymphocytes by adherence to plastic surface. Cells were incubated for 16 hours with and without endotoxin.TF activity was measured in the cells by modified PT and TF antigen by ELISA. TF activity and antigen were 1.4–1.7x 10−5 U/monocyte and 21–31x10−5 pg/monocyte in unstimulated PBMC from normal controls (NC), RT and ET. Stimulated PBMC from NC and RT showed a similar increase of TF activity and antigen (2.5 and 3.8 fold).However, PBMC from ET generated 12.9 and 14.4 times more TF activity and antigen (p

Description: Wnt signalling is known to play an important regulatory role in hematopoietic progenitors/stem cells during both fetal and adult development. Recently this pathway has been implicated in several hematological malignancies, however, its precise role in pathogenesis of pre-B ALL remains unknown. In this study we examined the expression of genes required for Wnt signalling in 12 ALL cases, normal bone marrow mononuclear cells, bone marrow stroma and the effect of Wnt-3a stimulation on leukemic cell growth and survival. RT-PCR analysis revealed expression of Wnt family members 2b, 5a, 10b and 16b in most ALL cases. Primary BM stroma expressed Wnt-5a alone while BM mononuclear cells expressed Wnts 3a, 5a, 10b and 16b. Wnt receptors Fz-7 and 8 were widely expressed on ALL cases while Fz-3 and 4 showed restricted expression. Together this suggests that Wnt signalling pathway may be active in pre-B ALL cells. To characterise functional consequences of Wnt pathway activation we investigated the effects of Wnt-3a on proliferation, survival and cell cycle status of blasts from three pre-B ALL cell lines, one newly derived stromal dependent cell line (STYG) and one primary patient sample under 3-day serum-free culture conditions. Exposure to Wnt-3a resulted in a mean 5.3-, 5.2-, 1.7-, 2.7- and 5.1- fold stimulation of 3H-thymidine incorporation in NALM6, Reh, LK63, STYG and primary ALL cells, respectively. Cell cycle analysis based on propidium iodide staining confirmed an increased proportion of cells progressing from G0/1 through to S/G2/M phases. Wnt3a augmented survival in all leukemic cells by 18.6% (range 11.7% – 24.6%, p

Description: Hox (homeobox) genes are known to be key regulators of development and haematopoiesis and several have also been implicated in leukaemogenesis. Overexpression of HoxA5 in human haematopoietic progenitors leads to an increase in myelopoiesis, suggesting a role for this hox gene during induction of myeloid differentiation. Inactivation of genes by CpG island DNA methylation is known to be important in the development and progression of leukaemia, and inhibitors of DNA methylation are currently of great interest as novel therapeutics for a number of haematopoietic malignancies. Here we show that in peripheral blood from healthy volunteers HoxA5 exhibits methylation of 50% of alleles across an extensive CpG island covering the promoter region/1st exon of HoxA5. In patients with chronic myeloid leukaemia, 33% (15/45 patients) exhibited increased methylation of HoxA5 (80–100% of alleles methylated) in the chronic phase of the disease. However, such hypermethylation of HoxA5 was invariably present in samples from patients in myeloid blast crisis (15/15 patients). In contrast, patients in lymphoid blast crisis did not exhibit increased levels of hypermethylation. Analysis of patients in chronic phase demonstrated a statistically significant correlation (p 〈 0.006) between hypermethylation of the HoxA5 gene and other prognostic factors associated with high risk of progression to blast crisis (high Hasford/Sokal score, incomplete response to Imatinib, known early progression, del 9), suggesting that methylation of HoxA5 may be a clinically useful prognostic indicator. The results are also compatible with a direct role for hypermethylation of HoxA5, and consequent loss of HoxA5 expression, in inhibition of myeloid differentiation during progression to blast crisis and implicate HoxA5 as a therapeutic target for inhibitors of DNA methylation in the treatment of leukaemia.

Description: Background: Over the last decade, increasing numbers of families with at least 2 cases of CLL or other B-cell lymphoproliferative disorders have been described. Methods: Families seen at Mayo Clinic with at least one individual diagnosed with CLL and one or more first or second degree relative diagnosed with CLL or a B-cell lymphoproliferative disorder(LPD) were identified. Clinical characteristics and risk stratification test results (cytogenetic abnormalities by FISH, level of CD38 expression, and IgVH gene mutation status) were extracted and reviewed. Serum, intracellular and CLL B cell secreted levels of pro- and anti-angiogenic cytokines (VEGF, BFGF, and TSP) were also compared between cases of familial and sporadic CLL. Results: Seventy-one families seen at Mayo Clinic were identified meeting criteria for familial CLL. Of the 71 index CLL families, 78.9% (n=56) had at least one other first or second-degree relative with CLL or a B cell LPD, 15.5% (n=11) had 2 other affected members, and 5.6% (n=4) had 3 or more affected members. Of affected family members with B-cell LPDs other than CLL, 64% (n=25) had non-Hodgkin lymphoma, 21% (n=8) had Hodgkin disease, 10% (n=4) had multiple myeloma or Waldenstrom macroglobulinemia, and 5% (n=2) had another lymphoid leukemia (1 ALL, 1 hairy cell leukemia). The median age was 62, with 63% males and 37% females. The majority of individuals had an early Rai stage at diagnosis (stage 0–66%, stage I-19%). Results of cytogenetic testing by FISH, CD38 status, IgVH gene mutation status appear similar in familial and sporadic CLL cases seen at Mayo clinic during the same time interval. No clear pattern of IgVH gene mutation status, IgVH gene family usage, level of CD38 expression, or cytogenetic abnormalities by FISH was seen among multiple affected members from the same family. There were also no significant differences noted in serum, intracellular, and CLL B cell secreted levels of VEGF, BFGF or TSP in the familial samples when compared to a cohort of non-familial samples. Conclusions: Expression of cytogenetics by FISH, immunophenotype, IgVH mutation status and levels of serum, intracellular and cytoplasmic pro- and anti-angiogenic factors(VEGF, BFGF, and TSP) were similar in individuals with familial and sporadic CLL.

Description: One of the most intriguing features of chronic lymphocytic leukemia (CLL) is its clinical heterogeneity. The mutational status of immunoglobulin heavy chain variable region (IgVH) genes is one of the most powerful predictors of overall survival (OS) and progression-free survival (PFS). The expression of the tyrosine kinase ZAP-70 in CLL cells has been proposed as a surrogate marker for the mutational status of IgVH genes. Recent reports suggest that ZAP-70 over-expression is an independent prognostic factor for shorter OS and PFS. There is little information about correlation between ZAP-70 expression and other clinical and biological features in CLL. The aims of this study are 1) to evaluate prospectively ZAP-70 expression in a series of CLL patients; 2) to correlate this with clinical stage, lymphocyte morphology, CD38 expression and chromosomal abnormalities detected by FISH and 3) to analyze the independent prognostic value of ZAP-70 in predicting time to first treatment (treatment free interval, TFI). ZAP-70 expression was analyzed using four-colour flow cytometry (as described by Crespo et al, NEJM2003; 348:1764–75). 201 previously untreated CLL patients were evaluated at diagnosis (32%) or during the disease course. Amongst them, 143 patients were entered in the LRF CLL4 trial. Clinical stage at the time of the study was A in 92 patients (A progressive in 46), B in 63 and C in 46. 28% were ZAP-70 positive (+) (cut-off ≥ 20% of CD5/CD19+ cells). ZAP-70 positivity was associated with prevalence of stage B/C at diagnosis (p=0.004), CLL with 〉10% prolymphocytes (CLL/PL) or atypical morphology (p

Description: Introduction: FL is generally responsive to conventional-dose chemotherapy but long term disease-free survival (DFS) is uncommon. High-dose chemo-radiotherapy followed by ASCT has the potential to induce remission in this disease but the long-term benefit of this modality remains to be determined. Methods: Between 1990 and 2003, we transplanted 52 pts originally diagnosed with low-grade FL (31 grade 1, 21 grade 2). Twenty-five (48%) had biopsy-proven large cell transformation (FL grade 3 or diffuse large cell lymphoma) before ASCT. The median number of prior therapies was 2 (range: 1 to 7). Prior to ASCT, 45 pts (87%) were responsive to salvage therapy with 20 pts (38%) in CR. Five pts (10%) had chemo-resistant disease at the time of ASCT. High-dose regimens included BCNU-cyclophosphamide-etoposide (31%), melphalan/TBI (27%), and cyclophosphamide/TBI (25%). Thirty-eight pts (73%) received peripheral stem cells (PSCT) and 14 pts (27%) received autologous bone marrow (BM) with 4-hydroxyperoxycyclophosphamide (4-hc) purging in 9 cases (17%). The median age was 49 yrs (range: 29–65). Results: There was 1 treatment-related death during the first 100 days. After ASCT, 36 pts (69%) achieved a CR, 2 (4%) had a PR, and 7 (13%) had stable disease. Among those in CR, 20 (56%) had a CR pre-ASCT, 14 (41%) had a lesser response, and 1 (3%) was chemo-resistant. Median follow-up (f/u) of survivors was 5.3 yrs (range: 1.7 months to 12.4 yrs). The median overall survival (OS) has not yet been reached. The median event-free survival (EFS) is 3.4 yrs (range: 1.7 months to 12.4 yrs). Among complete responders, more than 50% are disease free at last follow-up (range 1.7 months to 12.1 yrs). Variables favorably affecting EFS and OS are age 〈 60 yrs (p = 0.007, 0.015 respectively), achievement of a CR after ASCT (p = 0.002, 0.001), absence of transformation (p = 0.038, 0.017), BM vs. PSCT (p = 0.042, 0.086), and 4-hc BM purging (p = 0.044, 0.059). Number of prior regimens, response prior to ASCT, type of preparative regimen, and addition of TBI, were not significantly associated with EFS, DFS, or OS. In multivariable analysis, achievement of CR after ASCT and age 〈 60 yrs are the only significant predictors of EFS and OS. Adjusted for age, 53% of pts with a CR after ASCT are alive and event-free at last f/u (range: 2.4 months to 12.4 yrs) (Figure 1). In contrast, the median EFS among pts without a CR is 0.5 yrs (range: 1.7 months to 5.3 yrs). Conclusion: ASCT is a reasonable therapeutic approach to FL, resulting in long term EFS for some pts, even with relapsed, refractory and/or transformed disease. In our experience, significant predictors of EFS and OS after ASCT are complete response and age

Description: The cell of origin of childhood acute lymphoblastic leukaemia (ALL) has been the subject of conflicting reports in recent years. One model suggests that many haemopoietic cell types are susceptible to transformation and the level of commitment of the target cell influences the characteristics of the resulting blast cell population. A second model suggests that primitive haemopoietic cells are the targets for transformation, with some differentiation occurring subsequent to the transformation event. This model suggests a hierarchy of progenitors may exist in ALL. In support of this latter model, we have demonstrated that leukaemic stem cells in B-ALL have a primitive CD34+/CD10−/CD19− phenotype and T-ALL cells with NOD/SCID engrafting capacity are CD34+/CD4−. In this investigation we have attempted to further purify and characterise leukaemic stem cells from children with T-ALL. Cells from 7 patients were sorted for expression of CD34 and CD7 and the sorted subfractions evaluated for long-term proliferative ability in vitro using a serum free suspension culture assay and in the NOD/SCID mouse model. In this group of patients, the CD34+/CD7+ fraction represented 7±6% of cells at sorting, 6±4% were CD34+/CD7− and the majority were CD34−/CD7+ (60±12%). After 3 weeks in culture, the majority of proliferating cells were derived from the CD34+/CD7− subfraction (53±16%). By week 6, 〉70% of proliferating cells were derived from the CD34+/CD7− subfraction. Unsorted ALL cells and the sorted subfractions from 4 of these patients, were evaluated for their ability to engraft sublethally irradiated NOD/SCID mice. In each case, engraftment was achieved using 105–106 unsorted cells (25–80% CD45+) and with the CD34+/CD7− subfraction only (4–84% CD45+ with 3x103–8x104 cells). There was no engraftment with the other subfractions despite injecting up to 100 fold more cells. The engrafted cells had the same karyotype as the patient at diagnosis and expressed high levels of CD2, CD4 and CD7 implying they had differentiated in vivo. The self-renewal capacity of the CD34+/CD7− cells was evaluated by secondary transplantation. CD45+ cells from NOD/SCIDs engrafted with CD34+/CD7− cells successfully engrafted secondary recipients with equivalent levels of human cell engraftment, demonstrating these cells were capable of self-renewal. These findings suggest that cells with a more primitive phenotype may be the targets for transformation in T-ALL, rather than committed lymphocytes. To further investigate this hypothesis, we sorted cells from 4 of these patients for expression of CD133 and CD7 and evaluated their proliferative ability as described above. Results to date indicate that the CD133+/CD7− fraction represents only 0.35% of nucleated cells at sorting. However, after 3 weeks in culture, 48±9% of proliferating cells were derived from this subfraction and by week 6, 58±20% of cells were derived from the CD133+/CD7− subfraction. In vivo analyses completed in 2 patients to date have shown that only the CD133+/CD7− subfraction was capable of engrafting NOD/SCID mice (0.5–54% CD45+ using 3x103–105 cells). These results demonstrate that T-ALL cells with long-term proliferative and NOD/SCID repopulating capacity express the primitive haemopoietic cell antigens CD133 and CD34 and lack expression of T-lineage markers. These findings add further support to the concept of a common cell of origin for acute leukaemias.

Description: Several cytokines and growth factors are involved in the regulation of megakaryocytopoieses and platelet formation. Interleukin-11 (IL-11), IL-6, IL-3, IL-1b, and thrombopoietin (TPO) act synergistically to promote proliferation and maturation of megakaryocytes. Recombinant IL-11 and TPO are under clinical investigation as supplements to stimulate thrombopoiesis in patients with cancer. We investigated the plasma levels of TPO in 127 patients with chronic lymphocytic leukemia (CLL) and correlated these levels with platelet counts and various laboratory and clinical characteristics. TPO levels were significantly higher in patients with CLL (median, 232 pg/mL; range, 26.4–2714.5 pg/mL) than in healthy control subjects (P

Description: The present study was designed to evaluate prothrombotic risk profiles in 59 consecutively recruited white neonates with renal venous thrombosis (RVT). The rates of prothrombotic risk factors (PRs)—for example, the factor V (FV) 1691G〉 A mutation, the factor II (FII) 20210G〉 A variant, antithrombin (AT), protein C (PC), protein S (PS), elevated lipoprotein(a) (Lp(a)), total fasting plasma homocysteine (tHcy) levels, and anticardiolipin antibodies (ACAs)—were compared with those of 118 healthy control children. At onset, 32 (54.2%) of the 59 neonates showed underlying clinical conditions; 40 (67.8%) of them and 23 (85.2%) of the 27 infants with idiopathic RVT showed at least one PR. Univariate analysis revealed significantly elevated odds ratios/95% confidence intervals (ORs/95% CIs) for FV and Lp(a). Additionally, PC/AT deficiency and ACAs were found significantly more often in the patient group (P = .04). Multivariate analysis calculated significant ORs/95% CIs only for FV (OR, 9.4; 95% CI, 3.3-26.6) and elevated Lp(a) (OR, 7.6; 95% CI, 2.4-23.8). Of the 59 neonates investigated, 53 revealed renal atrophy, and 13 children additionally suffered from severe arterial hypertension. In conclusion, the present study demonstrates the significance of genetic PR—especially the FV mutation and elevated Lp(a)—for the etiology of neonatal RVT.

Description: The high frequency of Kaposi sarcoma (KS) in immunodeficiency states, particularly in patients with AIDS, has been attributed to increased replication of KS-associated herpesvirus (KSHV), a necessary cofactor for KS development. However, experimental KSHV infection of endothelial lineage cells that compose KS lesions has been difficult even in the absence of immune cells. Here we show that HIV-1 Tat protein can directly promote KSHV transmission. Full-length HIV-1 Tat and a 13–amino-acid peptide corresponding to the basic region of Tat specifically enhances the entry of KSHV into endothelial and other cells, presenting evidence for an active role of HIV-1 in the development of KSHV-associated diseases. These results can explain why AIDS-KS is more frequent and clinically more aggressive than KS in other immunodeficiency states.

Description: Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study. (Blood. 2004;103: 3465-3473)

Description: HIV-1- and cytomegalovirus (CMV)-specific CD4 T-cell-mediated antiviral immunity was evaluated by assessing the frequency of interleukin 2 (IL-2)- and interferon γ (IFN-γ)-secreting cells following antigen-specific stimulation in blood and lymph node. HIV-1-infected subjects with progressive disease at early stage of infection with no previous history of antiretroviral therapy (ART), subjects with nonprogressive disease, and HIV-negative subjects were studied. On the basis of the ability to secrete IL-2 and IFN-γ, 3 functionally distinct populations of CD4 T cells were identified: (1) IL-2-secreting cells; (2) IL-2/IFN-γ-secreting cells; and (3) IFN-γ-secreting cells. CMV-specific CD4 T cells were almost equally distributed within the 3 functionally distinct cell populations in the 3 study groups as well as HIV-1-specific CD4 T cells in subjects with nonprogressive disease. However, a skewing toward IFN-γ-secreting cells (70% of HIV-1-specific CD4 T cells) was observed in subjects with progressive disease, and IL-2- and IL-2/IFN-γ-secreting cells were almost absent. The frequencies of IL-2- and of IL-2/IFN-γ-secreting HIV-1-specific CD4 T cells were negatively correlated with the levels of viremia. Interestingly, prolonged ART was able to correct the skewed representation of different populations of HIV-1-specific CD4 T cells but was associated with only a partial recovery of IL-2-secreting cells. These results indicate that the composition of the pool of functionally distinct virus-specific CD4 T cells is important for virus control. (Blood. 2004;103:966-972)

Description: Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals. (Blood. 2004;103: 3412-3419)

Description: The degree of redundancy between thrombopoietin (Tpo) and steel factor (SF) cytokine pathways in the regulation of hematopoiesis was investigated by generating mice lacking both c-Mpl and fully functional c-Kit receptors. Double-mutant c-Mpl–/–KitWv/Wv mice exhibited reduced viability, making up only 2% of the offspring from c-Mpl–/–KitWv/+ intercrosses. The thrombocytopenia and megakaryocytopenia characteristic of c-Mpl–/– mice was unchanged in c-Mpl–/–KitWv/Wv mice. However, the number of megakaryocytic colony forming units (CFU-Mks) was significantly reduced, particularly in the spleen. While KitWv/Wv mice, but not c-Mpl–/– mice, are anemic, the anemia was more severe in double-mutant c-Mpl–/–KitWv/Wv mice, indicating redundancy between Tpo and SF in erythropoiesis. At the primitive cell level, c-Mpl–/– and KitWv/Wv mice have similar phenotypes, including reduced progenitors, colony forming units–spleen (CFU-Ss), and repopulating activities. All of these parameters were exacerbated in double-mutant mice. c-Mpl–/–KitWv/Wv mice had 8-fold fewer clonogenic progenitor cells and at least 28-fold fewer CFU-Ss. c-Mpl–/– mice also demonstrated a reduced threshold requirement for nonmyeloablative transplant repopulation, a trait previously associated only with KitW mice, and the level of nonmyeloablative engraftment was significantly greater in c-Mpl–/–KitWv/Wv double mutants. Thus, c-Mpl–/–KitWv/Wv mice reveal nonredundant and synergistic effects of Tpo and SF on primitive hematopoietic cells.

Description: The advent of therapeutic monoclonal antibodies has enhanced the efficacy of NHL treatment. In recent years, these immuno-therapies have been increasingly used in therapy. We conducted a population-based study of NHL treatment practices in the US using a stratified random sample of patients diagnosed in 1999 with histologically confirmed NHL (n=939) residing in the geographic areas covered by the Surveillance, Epidemiology and End Results program. Blacks and Hispanics were over-sampled to obtain more stable estimates. Patients were followed for vital status through Dec 2001. We performed separate logistic regression analyses to study the potential factors associated with the likelihood of receiving chemotherapy, radiation therapy and the monoclonal antibody, Rituximab. Cox Proportional Hazards regression model was used to study the risk factors associated with survival time. We grouped histological subtypes into five broad categories: B-cell aggressive, B-cell indolent, T-cell generic, cutaneous T-cell, and mantle cell lymphomas. The majority of patients presented with B-cell aggressive or B-cell indolent lymphomas (n=828). Approximately 20% of patients received no therapy. Over 60% of patients received chemotherapy, either alone or in combination. 12% of patients received Rituximab and it was most frequently administered to patients in combination with chemotherapy, especially for patients with B-cell aggressive, B-cell indolent and T-cell generic lymphomas. Only 3% of patients participated in clinical trials. Age and gender were associated with the receipt of chemotherapy: people aged over 75 years, and males were less likely to have received chemotherapy (P=0.01). There were no significant associations between the likelihood of receiving Rituximab and the demographic and clinical factors analyzed. However, our results suggested that African-Americans and people aged over 75 years were less likely to have received immunotherapy. Twenty-four percent of patients received radiation with or without another therapy. When compared to patients with no symptoms at presentation, patients who presented with B-symptoms at diagnosis or those whose B-symptoms were unknown were less likely to have received radiation therapy (OR=0.32 and 0.47 respectively, P=0.0002). Approximately 50% of patients had died by the end of maximum the 3-year follow-up period. Both cause-specific and all-cause mortality was significantly associated with patient age, race/ethnicity, gender, marital status and co-morbid conditions, as well as histological subgroup. Hispanic and Black patients had higher risk of death from both NHL and all-cause (P 75 years, male patients, unmarried patients, or patients with B-symptoms had higher risk of death from either NHL or all-cause (p

Description: The deregulation of the immune response is a critical component in inflammatory disease. Recent in vitro data show that T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of cytokine signaling. Furthermore, tc-ptp-/- mice display immune defects and die within 5 weeks of birth. We report here that tc-ptp-/- mice develop progressive systemic inflammatory disease as shown by chronic myocarditis, gastritis, nephritis, and sialadenitis as well as elevated serum interferon-γ. The widespread mononuclear cellular infiltrates correlate with exaggerated interferon-γ, tumor necrosis factor-α, interleukin-12, and nitric oxide production in vivo. Macrophages grown from tc-ptp-/- mice are inherently hypersensitive to lipopolysaccharide, which can also be detected in vivo as an increased susceptibility to endotoxic shock. These results identify T-cell protein tyrosine phosphatase as a key modulator of inflammatory signals and macrophage function. (Blood. 2004;103:3457-3464)

Description: HuM195, a humanized anti-CD33 monoclonal antibody, targets myeloid leukemia cells and has activity against minimal residual disease. To enhance the potency of native HuM195 and avoid nonspecific cytotoxicity seen with β-emitting radioimmunoconjugates, the α-emitting radiometal 213Bi was conjugated to HuM195. The feasibility, safety, and antileukemic activity of therapy with 213Bi-HuM195 were shown in a phase I trial (Jurcic et al. Blood 2002). Because of the short-range (50–80 μm) and high linear energy transfer (8400 keV) of α particles, radioimmunotherapy with 213Bi is ideally suited for the treatment of residual disease. To determine the effects of 213Bi-HuM195 after partial cytoreduction with chemotherapy, we treated 25 patients (median age, 67 years; range, 49–80) with cytarabine 200 mg/m2/day for 5 days followed by 213Bi-HuM195 in a phase I/II trial. Fourteen patients had relapsed or primary refractory AML; 5 patients had previously untreated de novo AML, and 6 patients had untreated secondary AML. Sixteen patients had intermediate-risk cytogenetics, and 9 had poor-risk cytogenetics. During the phase I portion of the study, cohorts of 3–6 patients were treated with 0.5, 0.75, 1, and 1.25 mCi/kg. At the 1.25 mCi/kg dose level, 2 of the 4 patients had dose-limiting myelosuppression (grade 4 leukopenia lasting ≥ 35 days) and 1 patient died of progressive pneumonia. Therefore, the maximum tolerated dose was determined to be 1 mCi/kg. No responses were seen at the first two dose levels. Seven of the 19 patients (37%) who received 1 mCi/kg (n=15) or 1.25 mCi/kg (n=4) responded. There were 2 CRs lasting 9 and 12 months; 3 CRp (CR with incomplete platelet recovery) lasting 1, 2, and 6 months and 2 PRs lasting 3 and 8 months. The median time from initiation of chemotherapy to recovery of leukocyte counts was 34 days (range, 21–59 days). Delayed count recovery was attributed to persistent leukemia in 6 patients. Neutropenic fever occurred in all patients, and 19 patients had documented infections. At the phase II dose level, two of the 15 patients died of progressive infections, and one had grade 4 hyperbilirubinemia. The most common extramedullary toxicities were transient, low-grade elevations in liver function tests (n=19) and serum creatinine (n=11). Thirteen patients had infusion-related reactions following the first injection of 213Bi-HuM195, typically characterized by reversible grade 1 or 2 fever and/or chills. One patient had orthostatic hypotension and syncope after the first antibody infusion associated with concomitant bacteremia. Sequential administration of cytarabine and 213Bi-HuM195 is tolerable and can produce complete remissions in patients with AML.

Description: The acquired genetic characteristics of acute lymphoblastic leukemia (ALL) blasts are often used to guide the intensity of therapy, whereas the germline host genetic characteristics of the patient generally have not been considered. Multiple common, functionally important polymorphisms affect genes whose products determine the pharmacokinetics and pharmacodynamics of antileukemic agents. It is not yet known how genetic polymorphisms may interact to affect the outcome of antileukemic therapy. Combining classification and regression tree with failure time analysis, we assessed whether 16 genetic polymorphisms, alone or in combination, predicted relapses in 246 children with ALL, 116 of whom were treated on the lower-risk (LR) and130 on the higher-risk (HR) arms of the St Jude protocol Total XIIIB. Genotyping was performed for the following polymorphic loci: CYP3A4*1B and CYP3A5*3; GSTP1 313A〉G, GSTM1 and GSTT1 deletions; MDR1 exon 21 (2677G〉T/A) and MDR1 exon 26 (3435C〉T); MTHFR 677C〉T and MTHFR 1298A〉C; NR3C1 1088A〉G; SLC19A1 80G〉A; TPMT 238G〉C, 460G〉A and 719A〉G; TYMS enhancer repeat; UGT1A1 promoter repeat polymorphism; VDR intron 8 G〉A and VDR FokI (start-site) T〉C. In all children with available RNA in their diagnostic ALL blasts, gene expression levels of prognostic genotypes were analyzed using the Affymetrix genechip array HG_U95Av2. Among the HR group, the glutathione S-transferase M1 (GSTM1) non-null genotype was associated with the risk of hematological relapse (5-year cumulative incidence, 17.1%±4.5% compared to 5.1%±2.9% for GSTM1 null genotype, p = 0.03), and among the non-null genotypes, the thymidylate synthetase (TYMS) 3/3 genotype was associated with a further increase in hematologic relapse risk (5-year cumulative incidence, 29.2%±9.5% compared to 10.9%±4.7% for TYMS 2/3 or 2/2 genotypes, p = 0.02). Increased expression levels of these two target genes (p 〈 0.0001 and p = 0.09, respectively) were consistent with resistance to the drugs interacting with these gene products. For central nervous system relapse, among the HR group, the vitamin D receptor (VDR) start site (p = 0.02) and intron 8 genotypes (p = 0.04) predisposed, whereas for LR patients the TYMS 3/3 genotype predisposed (p = 0.04). The genotypes associated with outcome have pharmacologic plausibility: e.g., high GST activity (GSTM1 non-null) could cause anticancer drug resistance; high TYMS activity (TYMS 3/3) would be less inhibited by antifolates. In conclusion, germline polymorphisms influence the outcome of antileukemic therapy, and therefore represent determinants of response that can be used to optimize therapy.

Description: Background: Little is known about the role of the CD56+ natural killer (NK) cell dose on the outcome of allogeneic peripheral blood stem cell transplantation (PBSCT). Recently, a higher dose of NK cells has been associated with a lower incidence of severe GVHD in a PBSCT setting. Therefore, the current study attempted to evaluate the effect of the NK cell dose on transplant outcomes, including non-relapse mortality (NRM) and infectious events, in an allogeneic PBSCT setting. Methods and Materials: Sixty-one cytokine mobilized PBSC recipients from HLA-matched sibling donors were analyzed according to the infused dose of CD34+ cells and NK cells in relation to overall survival (OS), NRM, GVHD, and infectious events. Results: The group of patients that received a higher dose of NK cells (≥ 5x107/Kg) showed a lower incidence of NRM (p=0.0186) and infectious events (p=0.0107). When confining the analysis to the group that received a CD34+ cell dose of ≥ 6x106/Kg, those patients that received a higher dose of NK cells exhibited a lower incidence of extensive chronic GVHD (p=0.0704). In a multivariate analysis using Cox’s regression model, a higher dose of NK cells was significantly associated with better transplant outcomes (for NRM, NK cell dose p=0.042, for CD34+ cell dose p=0.018; for infectious events, NK cell dose p=0.013, CD34+ cell dose 0.016; for bacterial infection, NK cell dose p=0.049). The group that received a higher NK cell dose also showed a faster immune recovery (p=0.046 for NK cell recovery, p=0.034 for helper T-cell recovery) in serial measurements of peripheral lymphocyte subsets at D+90, +180, and +365. Conclusions: The present data suggests that a high dose of NK cells may play an important role in improving transplant outcomes, in terms of reducing NRM and infectious events together with CD34+ cells. The protective role of NK cells against infections may also be associated with a faster immune recovery after allogeneic PBSCT. Figure Figure Figure Figure

Description: Autologous stem cell transplant (ASCT) requires therapy with very highly emetogenic chemotherapy regimens. Nausea and emesis are a major issue for patients undergoing ASCT. Aprepitant, a selective substance-P/neurokinin 1 (NK1) receptor antagonist, has been approved by the U.S. Food and Drug Administration for treatment of acute and delayed CINV in combination with a corticosteroid and 5-HT3 receptor antagonist. Due to little patient data in this area, a retrospective chart review was performed which evaluated 41 ASCT patients that had received standard therapy (control group, n=25) compared to aprepitant in addition to standard therapy (aprepitant group, n=16). Patients who underwent an ASCT and were greater than 18 years of age were eligible for inclusion in the study. Patients who underwent an allogeneic stem cell transplant were excluded. All data was collected from the hospital’s computerized nurse charting system. Episodes of nausea and emesis were collected from nursing notes while data for rescue medications was collected from the charted medications. Conditioning regimens given during the study included high dose (HD) melphalan, BEAM (carmustine, etoposide, cytarabine, and melphalan) or R-BEAM (rituximab, carmustine, etoposide, cytarabine, and melphalan). All patients evaluated in the aprepitant group had received aprepitant 125 mg orally once daily on the first day of chemotherapy administration and 80 mg orally once daily the following 2 days in the HD melphalan group (Day -1 through Day 0) and the following 5 days in the BEAM group (Day-6 through Day -1). Although aprepitant dosing was consistent, dosing was determined by the individual physician and prescribed on a patient-specific basis. The primary endpoint was the number of episodes of nausea and emesis after chemotherapy, while the secondary endpoint was the number of rescue medications that were used for nausea and emesis. For the primary endpoint of the study, the mean number of nausea episodes per patient was reduced in the aprepitant group compared to the control group in the acute phase (1.7 versus 2.2, p=0.51) and delayed phase (5.3 versus 5.6, p=0.83). Emetic episodes per patient were decreased in the aprepitant group compared to the control group in both the acute (0.06 versus 0.9, p=0.21) and delayed phases (0.9 versus 2.4, p=0.17). Use of rescue medication per patient was decreased in the aprepitant group compared to the control group in the acute phase (2.3 versus 3.6, p=0.40) but not in the delayed phases (8.1 versus 8.1, p=0.99). None of the results of this study were statistically significant, although the reduced number of emetic episodes with aprepitant may be clinically significant. The results of this study warrant consideration for a prospective evaluation of this therapy in ASCT patients to greater clarify the use of aprepitant in these patients.

Description: The physiologic significance of MHC-peptide complex presentation by endothelial cells (ECs) to trafficking T lymphocytes remains unresolved. On the basis of our observation that cognate recognition of ECs enhanced transendothelial migration of antigen-specific T lymphocytes in vitro, we have proposed that by displaying antigenic peptides from the underlying tissue, ECs promote the recruitment of antigen-specific T cells. In this study, we have tested this hypothesis by comparing the trafficking of HY-specific T lymphocytes into antigenic and nonantigenic tissue using in vivo models of T-cell recruitment. Up-regulated expression of H2 molecules presenting endogenous antigen in the peritoneal mesothelium and vessels led to the local recruitment of HY-specific T cells in male, but not female, mice. Intravital microscopy experiments analyzing EC–HY-specific T-cell interactions in the cremasteric vascular bed revealed that cognate recognition of the endothelium results in enhanced diapedesis of T cells into the tissue, while not affecting rolling and adhesion. Our results are consistent with the hypothesis that, under inflammatory conditions, antigen presentation by the endothelium contributes to the development and specificity of T-cell–mediated inflammation by favoring the selective migration of antigen-specific T cells. (Blood. 2004;103:3111-3116)

Description: Mutations of hepcidin (HAMP) and hemo-juvelin (HJV) genes have been recently demonstrated to result in juvenile hemochromatosis. Expression of HAMP is regulated by iron status or infection, whereas regulation of HJV is yet unknown. Using quantitative real-time polymerase chain reaction, we compared expression of Hamp and Rgmc (the murine ortholog of HJV) in livers of mice treated with iron, erythropoietin, or lipopolysaccharide (LPS), as well as during fetal and postnatal development. Iron overload increased Hamp expression without effect on Rgmc mRNA. Erythropoietin decreased Hamp mRNA, but Rgmc expression was unchanged. Hamp mRNA level decreased after birth by 4 orders of magnitude, without significant changes in Rgmc expression. Administration of LPS elevated Hamp mRNA levels, while markedly decreasing hepatic Rgmc mRNA levels (to ∼5% after 6 hours). The responses of Hamp and Rgmc were quite different and suggested that human HJV expression could be modulated by inflammation.

Description: Background: Single autologous stem cell transplant (ASCT) is considered the standard of care for younger multiple myeloma (MM) patients (pts). However, it is not curative and virtually all patients will ultimately relapse. The role of a second ASCT as salvage therapy is unclear. Method: Retrospective review of all MM pts who received a 2nd ASCT as salvage therapy at Princess Margaret Hospital. Results: Between March 1992 and August 2004, 28 MM pts received a second ASCT for relapsed MM at our institution. Median age was 57 years (range 39–69) at second transplant. 17 pts were male. Immunoglobulin subtype included IgG (16), IgA (8), light chain (1), nonsecretory (2) and IgM(1). Median initial albumin was 42g/l (27–48). In 15 patients in whom cytogenetic studies were available, 2 were positive for the 13q deletion. Transplant conditioning regimen for first transplant was melphalan (MEL) + TBI +/− etoposide (E) in 6, MEL alone in 15 and other regimens in 7 pts. 2nd ASCT conditioning consisted of MEL + TBI +/− E in 2, MEL alone in 25 and BU+CY in 1. Median CD34 counts were 10.96x106/L and 4.85x106/L for 1st and 2nd ASCT respectively. The median time from diagnosis to first transplant was 9 months (2–74). The median time to relapse after the first transplant was 29 months (6–85), with a median interval between transplants of 39 months (6–99). The median time to progression after the second transplant was 13 months (5–56). No transplant-related deaths occurred. At median follow-up after 2nd ASCT of 15 months (1–60), 20 (71%) pts are alive. Twelve pts (43% of all pts) are free of disease progression. The 5-year actuarial progression-free survival (PFS) and overall survival (OS) was 47% and 32%, respectively. Long term progression-free status based on the progression-free interval after 1st transplant is summarized in the following table: Interval Total number of pts Number of progression-free pts ≤ 12 months 1 0 (0%) 12–24 months 9 3 (33%) ≤ 24 months 18 9 (50%) Conclusions: 2nd ASCT is a feasible and safe salvage therapy in relapsed MM patients; 2nd ASCT is effective in providing prolonged remission over one year; 2nd ASCT is most effective in patients whose time to progression after 1st ASCT exceeds 24 months.

Description: Polymorphonuclear leukocytes (PMNs) migrate from the blood into areas of inflammation by binding to the endothelial cells of blood vessels via adhesion molecules. Vascular adhesion protein-1 (VAP-1) is one of the molecules mediating leukocyte-endothelial cell interactions. It is also an endothelial cell-surface enzyme (amine oxidase) that produces reactive oxygen species during the catalytic reaction. To study the role of the enzymatic activity of VAP-1 in PMN extravasation, we used an enzymatically inactive VAP-1 mutant, specific amine oxidase inhibitors (including a novel small molecule compound), and anti-VAP-1 antibodies in several flow-dependent models. The enzyme inhibitors diminished PMN rolling on and transmigration through human endothelial cells under conditions of laminar shear stress in vitro. Notably, the enzyme inactivating point mutation abolished the capacity of VAP-1 to mediate transmigration. Moreover, the new VAP-1 inhibitor effectively prevented the extravasation of PMNs in an animal model of inflammation. These data show that the oxidase activity of VAP-1 controls PMN exit from the blood during the relatively poorly understood transmigration step. (Blood. 2004;103:3388-3395)

Description: Mesenchymal stem cells (MSC) mostly surround the vasculature system of bone marrow (BM). MSC have been shown to exhibit immune suppressive properties. Since MSC express MHC Class II antigen, the question is whether these cells can act as APC. To this end, we hypothesize that MSC have the ability to present non-self antigens while acting as immune modulators. These dual roles of MSC prevent exacerbated inflammatory responses in the BM, thereby preventing hematopoietic dysfunction. A ‘dampened’ immune response in BM during insults by foreign agents could cause protection of the barrier that separates BM cavity with the periphery. The phagocytic role of MSC was shown by confocal microscopy and fluoresbrite plain YG 1.0-micron microspheres. APC property was demonstrated by challenging MSC with C. albicans (pulsed MSC), followed by exposure to CD4+ cells. The latter was obtained by immunoselection from peripheral blood mononuclear cells (PBMC) cultured for 5 days with C. albicans (10 mg/ml). Proliferation of the CD4+ cells (3H-thymidine incorporation and cell counts) proved APC properties of MSC, at efficiency comparable to macrophages. Overall, the studies show that the window between APC function and the period at which MSC could become immune suppressive is critical, since activated T-cells could destroy the endothelial barrier between BM and lymphatics/peripheral circulation. These studies show that MSC could be key cells in regulating immune responses in BM, and thereby protect BM from failure.

Description: CD4+CD25+ T-regulatory (Treg) cells have been shown to critically regulate self- and allograft tolerance in several model systems. Studies of human Treg cells have been restricted by the small number present in peripheral blood and their naturally hypoproliferative state. To better characterize Treg suppressor cell function, we determined methods for the isolation and expansion of these cells. Stringent magnetic microbead-based purification was required for potent suppressor cell line generation. Culture stimulation with cell-sized Dynabeads coated with anti-CD3 and anti-CD28 monoclonal antibodies, CD4+ feeder cells, and interleukin 2, provided for marked expansion in cell number (100-fold), with retention and enhancement of suppressor function. The potent Treg cell lines suppressed proliferation in dendritic cell-driven allo-mixed lymphocyte reaction (MLR) cultures by more than 90%. The Treg-derived suppressor cells functioned early in allo-MLR because expression of activation antigens and accumulation of cytokines was nearly completely prevented. Importantly, cultured Treg cells also suppressed activated and matured dendritic cell-driven responses. These results demonstrate that short-term suppressor cell lines can be generated, and they can express a very potent suppressive activity. This approach will enable more detailed biologic studies of Treg cells and facilitate the evaluation of cultured Treg cells as a novel form of immunosuppressive therapy. (Blood. 2004;104:453-461)

Description: The present study demonstrates that CD4+CD25+ T cells, expanded in peripheral blood of HIV-infected patients receiving highly active antiretroviral therapy (HAART), exhibit phenotypic, molecular, and functional characteristics of regulatory T cells. The majority of peripheral CD4+CD25+ T cells from HIV-infected patients expressed a memory phenotype. They were found to constitutively express transcription factor forkhead box P3 (Foxp3) messengers. CD4+CD25+ T cells weakly proliferated to immobilized anti-CD3 monoclonal antibody (mAb) and addition of soluble anti-CD28 mAb significantly increased proliferation. In contrast to CD4+CD25– T cells, CD4+CD25+ T cells from HIV-infected patients did not proliferate in response to recall antigens and to p24 protein. The proliferative capacity of CD4 T cells to tuberculin, cytomegalovirus (CMV), and p24 significantly increased following depletion of CD4+CD25+ T cells. Furthermore, addition of increasing numbers of CD4+CD25+ T cells resulted in a dose-dependent inhibition of CD4+CD25– T-cell proliferation to tuberculin and p24. CD4+CD25+ T cells responded specifically to p24 antigen stimulation by expressing transforming growth factor β (TGF-β) and interleukin 10 (IL-10), thus indicating the presence of p24-specific CD4+ T cells among the CD4+CD25+ T-cell subset. Suppressive activity was not dependent on the secretion of TGF-β or IL-10. Taken together, our results suggest that persistence of HIV antigens might trigger the expansion of CD4+CD25+ regulatory T cells, which might induce a tolerance to HIV in vivo.

Description: Interleukin 6 (IL-6) is a growth and survival factor for multiple myeloma cells. As we report here, the IL-6–dependent human myeloma cell line INA-6 responds with a remarkably rapid and complete apoptosis to cytokine withdrawal. Among the antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of apoptosis regulators, only myeloid cell factor-1 (Mcl-1) was slightly induced by IL-6. Overexpression studies demonstrated, however, that IL-6 does not exert its survival effect primarily through this pathway. The IL-6 signal transduction pathways required for survival and the target genes controlled by them were analyzed by using mutated receptor chimeras. The activation of signal transducer and activator of transcription 3 (Stat3) turned out to be obligatory for the survival of INA-6 cells. The same held true for survival and growth of XG-1 myeloma cells. Gene expression profiling of INA-6 cells by using oligonucleotide microarrays revealed many novel IL-6 target genes, among them several genes coding for transcriptional regulators involved in B-lymphocyte differentiation as well as for growth factors and receptors potentially implicated in autocrine or paracrine growth control. Regulation of most IL-6 target genes required the activation of Stat3, underscoring its central role for IL-6 signal transduction. Taken together, our data provide evidence for the existence of an as yet unknown Stat3-dependent survival pathway in myeloma cells.

Description: Between November 1999 and August 2002, consenting adult elective cardiac surgery patients at Oregon Health & Science University, Portland Veteran’s Administration Medical Center, and St. Vincent’s Hospital who were undergoing cardiopulmonary bypass (CPB) were randomized at admission to receive either prestorage leukoreduced red cells (PSL-RBCs) or standard red cells (S-RBCs) in a prospective double-blind fashion. Only data from those transfused were analyzed. Outcome measures included death at 60 days, 60 day infection rate, and length of hospital stay (LOS). Patients at all 3 institutions were operated on by the same group of cardiovascular surgeons. Given higher baseline infection rates for coronary artery bypass grafts (CABG) randomization was stratified by CABG vs valve replacement (VR). All RBCs were issued with blinding hoods. All platelet transfusion were prestorage leukoreduced. RBC transfusion rates were 30% for CABG, 38 % for VR, and 63% for CABG + VR. Infections were determined by infection control nurses using standardized Centers for Disease Control criteria from hospital surveillance and records and follow-up phone calls. Deaths were determined from hospital records and follow-up calls, and verified by National Death Index data. The PSL-RBC arm included 304 patients and the S-RBC arm 258 patients. The two groups were well-matched demographically and by cardiovascular risk factors. Intent-to-treat analysis showed a 60 day mortality of 9.7% in the S-RBC arm and of 4.9% in the PSL-RBC arm (p=0.029). Heart failure as the sentinel cause of death accounted for most of the difference (45.5% of deaths in the S-RBC group vs 13.3% in the PSL-LR group). Death rates were procedure specific: CABG alone 〉 CABG + VR 〉 VR alone. There was no significant difference between the S-RBC and PSL-RBC groups with regard to overall infection rate at 60 days. Most infections were superficial wound infections in the CABG patients; however groups did not differ in more serious infections such as bacteremia (p=0.369) or pneumonia (p=0.360). There was no significant difference between the groups with respect to LOS exclusive of in-hospital deaths. Our results essentially replicate in a North American context those of a previous European trial (Van de Watering et al Circulation1998; 97:562) involving elective cardiac surgery patients undergoing CABG and/or VR surgery randomized to receive S-RBCs prepared by the European buffy coat method vs leukoreduced RBCs. Despite technical differences in RBC preparation, the excess deaths in both studies in the S-RBC group vs the leukoreduced group suggests that elective cardiac surgery patients undergoing CPB constitute an at-risk group both in the US and Europe which may benefit from use of PSL-RBC. The significance of transfusion-related immunomodulation (TRIM) in man has been the subject of intense controversy. Interestingly the cause of the increased mortality in the S-RBC group, both in this study and the European study, could not be explained by differences in infection rates. Given the preponderance of deaths in the CABG patients it is tempting to speculate this may reflect an interaction between residual passenger leukocytes and ischemia which is independent of the TH1/TH2 lymphocyte shift postulated to underlie TRIM.

Description: Thrombosis is a known risk in pediatric patients with leukemia. This risk is increased when L-asparaginase is administered. However, children with cancer may have thrombotic complications similar to adults even in the absence of L-asparaginase. The risk may be related to the presence of central lines, surgery, immobilization, or inherited thrombophilia. Cancer in adult patients is also associated with an increased risk of thrombosis that may be related to the disease itself. Low molecular weight heparin such as enoxaparin has become widely used in adult patients with thrombosis. However, there is little data regarding the use of enoxaparin in children undergoing myelosuppressive therapy for malignancies. The purpose of this study was to review the utilization of low molecular weight heparin, enoxaparin (Lovenox), in children with cancer at our institution who had thrombosis while undergoing myelosuppressive chemotherapy. In particular we were interested in the efficacy of enoxaparin in these patients, and if these children were able to continue their chemotherapy without adjustment or interruption secondary to bleeding complications. We conducted a retrospective review from 1999 through April 1, 2004 which yielded seven patients with malignancies and a vascular thrombotic event. The age range was 4–17 years. Diagnosis include: B-precursor ALL (n=3), T-ALL, Hodgkin’s disease, Anaplastic large cell lymphoma, and rhabdomyosarcoma (n=1 each). Six patients developed a deep vein thrombus or clot of the vena cava. One of these 6 patients also had a pulmonary embolus. One patient presented with manifestations of a unilateral cerebral vascular accident without evidence of a DVT. U/S and CT/MRI were performed on patients when appropriate. All patients were screened for Protein C & S deficiency, ATIII deficiency, Factor V Leiden mutation, prothrombin 20210a mutation, and lupus anticoagulant. Treatment was enoxaparin, 1–1.5 mg/kg/dose twice daily to maintain a heparin anti-Xa level of 0.5-1.5 IU/mL. Once the clot had resolved, the enoxaparin was maintained daily for a total of 3–6 months of therapy. All patients had resolution of their thrombosis within 1–2 months of initiation of enoxaparin, and none required delays or dose-reduction of their chemotherapy regimens while on anti-coagulation. There were 10 documented occurences of thrombocytopenia (platelet count 〈 50,000) in 2 patients without bleeding complications. There was a minimum of 50 days of documented thrombocytopenia while on enoxaparin. We conclude that enoxaparin is effective and safe treatment for thrombotic complications in children undergoing cancer chemotherapy.

Description: Lymphocyte Peyer patch adhesion molecule (LPAM) or α4β7 integrin is expressed on lymphocytes and is responsible for T-cell homing into gut-associated lymphoid tissues through its binding to mucosal addressin cell adhesion molecule (MAdCAM), which is present on high endothelial venules of mucosal lymphoid organs. We found in murine allogeneic bone marrow transplantation (BMT) models that recipients of α4β7– donor T cells had significantly less graft-versus-host disease (GVHD) morbidity and mortality compared with recipients of α4β7+ donor T cells. A kinetic posttransplantation analysis of lymphocytes in the intestines and mesenteric lymph nodes demonstrated a delayed invasion of lower numbers of α4β7+ T cells in recipients of α4β7– T cells compared with recipients of α4β7+ T cells. Histopathologic analysis of GVHD target organs revealed that recipients of α4β7– T cells developed less GVHD of the intestines and liver, whereas there was no difference in cutaneous and thymic GVHD between recipients of α4β7– or α4β7+ T cells. Finally, we found that in vivo GVT activity of α4β7– donor T cells was preserved. We conclude that the α4β7 integrin is important for the invasion of alloreactive donor T cells into the gut and the subsequent development of intestinal GVHD and overall GVHD morbidity and mortality.

Description: Two distinct types of CpG oligodeoxynucleotide (ODN) have been identified that differ in their capacity to stimulate antigen-presenting cells: CpG-A induces high amounts of interferon-α (IFN-α) and IFN-β in plasmacytoid dendritic cells (PDCs), whereas CpG-B induces PDC maturation and is a potent activator of B cells but stimulates only small amounts of IFN-α and IFN-β. Here we examined the ability of these CpG ODNs to enhance peptide-specific CD8+ T-cell responses in human peripheral blood mononuclear cells (PBMCs). The frequency of influenza matrix–specific “memory” CD8+ T cells was increased by both types of CpG ODN, whereas the frequency of Melan-A specific “naive” CD8+ T cells increased on stimulation with CpG-B but not with CpG-A. The presence of PDCs in PBMCs was required for this CpG ODN-mediated effect. The expanded cells were cytotoxic and produced IFN-γ on peptide restimulation. Soluble factors induced by CpG-A but not CpG-B increased the granzyme-B content and cytotoxicity of established CD8+ T-cell clones, each of which was IFN-α/-β dependent. In conclusion, CpG-B seems to be superior for priming CD8+ T-cell responses, and CpG-A selectively enhances memory CD8+ T-cell responses and induces cytotoxicity. These results demonstrate distinct functional properties of CpG-A and CpG-B with regard to CD8 T cells.

Description: The chemokine receptor CXCR4 and its functional ligand, CXCL12, are essential regulators of development and homeostasis of hematopoietic and lymphoid organs. Heterozygous truncating mutations in the CXCR4 intracellular tail cause a rare genetic disease known as WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), whose pathophysiology remains unclear. We report CXCR4 function in 3 patients with WHIM syndrome carrying heterozygous truncating mutations of CXCR4. We show that CXCR4 gene mutations in WHIM patients do not affect cell surface expression of the chemokine receptor and its internalization upon stimulation with CXCL12. Moreover, no significant differences in calcium mobilization in response to CXCL12 are found. However, the chemotactic response of both polymorphonuclear cells and T lymphocytes in response to CXCL12 is increased. Furthermore, immunophenotypic analysis of circulating T and B lymphocytes reveals a decreased number of memory B cells and of naive T cells and an accumulation of effector memory T cells associated with a restricted T-cell repertoire. Based on our results, we suggest that the altered leukocyte response to CXCL12 may account for the pathologic retention of mature polymorphonuclear cells in the bone marrow (myelokathexis) and for an altered lymphocyte trafficking, which may cause the immunophenotyping abnormalities observed in WHIM patients. (Blood. 2004;104:444-452)